Administration of agents for the treatment of inflammation

ABSTRACT

A method of chronically reducing a patient&#39;s pathological inflammation via the administration of an agent that specifically binds to an alpha-4 integrin or a dimer comprising an alpha-4 integrin is disclosed. The agent provided must have a binding affinity such that administration is sufficient to suppress pathological inflammation, and the agent is administered chronically to provide long-term suppression of pathological inflammation.

This application is a continuation of U.S. Ser. No. 15/586,724, filedMay 4, 2017, now abandoned, which is a continuation of U.S. applicationSer. No. 14/613,821, filed Feb. 4, 2015, now abandoned, which is acontinuation of U.S. application Ser. No. 12/846,350 filed Jul. 29,2010, now abandoned, which is a divisional of U.S. application Ser. No.11/540,640, filed Oct. 2, 2006, now U.S. Pat. No. 7,807,167, which is adivisional of U.S. application Ser. No. 10/372,111, filed Feb. 25, 2003,now abandoned, which claims priority under 35 U.S.C. § 119 to U.S.Provisional Application Ser. No. 60/374,501 entitled ADMINISTRATION OFAGENTS FOR THE TREATMENT OF INFLAMMATION filed Apr. 23, 2002, andProvisional Application No. 60/360,134, entitled ADMINISTRATION OFAGENTS FOR THE TREATMENT OF INFLAMMATION filed on Feb. 25, 2002, theentire contents of each of which are hereby incorporated by reference intheir entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Jan. 20, 2020, is namedP1070US008_SL.txt and is 2,883 bytes in size.

FIELD OF THE INVENTION

This invention relates generally to agents that specifically bind to andinhibit an integrin receptor comprising an alpha-4 (α4) subunit, andtherapeutic uses of the same.

BACKGROUND OF THE INVENTION

Inflammation is a response of vascularized tissues to infection orinjury and is affected by adhesion of leukocytes to the endothelialcells of blood vessels and their infiltration into the surroundingtissues. In normal inflammation, the infiltrating leukocytes releasetoxic mediators to kill invading organisms, phagocytize debris and deadcells, and play a role in tissue repair and the immune response.However, in pathological inflammation, infiltrating leukocytes areover-responsive and can cause serious or fatal damage. See, e.g.,Hickey, Psychoneuroimmunology II (Academic Press 1990).

The integrins are a family of cell-surface glycoproteins involved incell-adhesion, immune cell migration and activation. Alpha-4 integrin isexpressed by all circulating leukocytes except neutrophils, and formsheterodimeric receptors in conjunction with either the beta1 or beta7integrin subunits; both alpha-4 beta-1 (α4β1) and alpha-4 beta-7 (α4β7)dimers play a role in the migration of leukocytes across the vascularendothelium (Springer et al., 1994 Cell 76: 301-14; and Butcher et al.,1996 Science 272: 60-6) and contribute to cell activation and survivalwithin the parenchyma (Damle et al., 1993 J. Immunol. 151: 2368-79;Koopman et al., 1994 J. Immunol. 152: 3760-7; and Leussink et al., 2002Acta Neuropathol. 103:131-136).

Specifically, alpha-4 beta-1 (also known as very late antigen-4(VLA-4)), binds to vascular cell adhesion molecule-1 (VCAM-1)(Lobb etal., 19941 J. Clin. Invest. 94:1722-8), which is expressed by thevascular endothelium at many sites of chronic inflammation (Bevilacquaet al., 1993 Annu. Rev. Immunol. 11: 767-804; and Postigo et al., 1993Res. Immunol. 144:723-35). The alpha-4 beta-7 dimer interacts withmucosal addressin cell adhesion molecule (MAdCAM-1), and mediates homingof lymphocytes to the gut (Farstad et al., 1997 Am. J. Pathol. 150:187-99; and Issekutz et al., 1991 J. Immunol. 147: 4178-84). Expressionof MAdCAM-1 on the vascular endothelium is also increased at sites ofinflammation in the intestinal tract of patients with inflammatory boweldisease (IBD) (Briskin et al., 1997 Am. J. Pathol. 151: 97-110).

Adhesion molecules such as alpha-4 integrins are potential targets fortherapeutic agents. For instance, the VLA-4 receptor, of which alpha-4integrin is a subunit, is an important target because of its interactionwith a ligand residing on brain endothelial cells. Diseases andconditions resulting from brain inflammation have particularly severeconsequences. In another example, the alpha-4 beta-7 integrin dimer isan important target due to its involvement in lymphocyte homing andpathological inflammation in the gastrointestinal tract.

Alpha-4 beta-1 integrin is expressed on the extracellular surface ofactivated lymphocytes and monocytes, which have been implicated in thepathogenesis of acute inflammatory brain lesions and blood brain barrier(BBB) breakdown associated with multiple sclerosis (MS) (Coles et al.,1999 Ann. Neurol. 46(3): 296-304). Agents against alpha-4 integrin havebeen tested for their anti-inflammatory potential both in vitro and invivo in animal models. See Yednock et al., 1992 Nature 356: 63-66; U.S.Pat. No. 5,840,299 issued to Bendig, et al. on Nov. 24, 1998, and U.S.Pat. No. 6,001,809 issued to Thorsett et al. on Dec. 14, 1999. The invitro experiments demonstrate that alpha-4 integrin antibodies blockattachment of lymphocytes to brain endothelial cells. Experimentstesting the effect of alpha-4 integrin antibodies on animals having anartificially induced condition simulating multiple sclerosis,experimental autoimmune encephalomyelitis (EAE), have demonstrated thatadministration of anti-alpha-4 integrin antibodies prevents inflammationof the brain and subsequent paralysis in the animals. Collectively,these experiments identify anti-alpha-4 integrin antibodies aspotentially useful therapeutic agents for treating multiple sclerosisand other inflammatory diseases and disorders.

In another specific example of pathological inflammation involvingalpha-4 integrins, Crohn's disease (CD) is a chronic, incurable,relapsing, transmural inflammation of the intestinal tract. The diseaseis characterized by inappropriate immune cell migration and activationin the intestinal mucosa involving T cells, macrophages and neutrophils(Schreiber et al., 1991 Gastroenterology 101: 1020-30). First-linemedical therapies for Crohn's disease include 5-aminosalicylates(5-ASAs), which have low efficacy, and corticosteroids, which havevarious short- and long-term side effects (Munkholm et al., 1994 Gut 35:360-2). Patients refractory to first-line therapies are treated withimmunosuppressive agents such as azathioprine, 6-mercaptopurine, andmethotrexate, but these agents have a slow onset of action andpotentially serious side effects (Stein et al., 2001 Surg. Clin. NorthAm. 81: 71-101, viii). More recently, biologic agents with a fasteronset of action have been introduced for use in treating Crohn'sdisease, but such agents are similarly plagued by issues such aslong-term efficacy and side effects.

SUMMARY OF THE INVENTION

The present invention provides methods of chronically reducingpathological inflammation in a patient via the chronic administration ofan agent that selectively binds to alpha-4 integrin. The chronic dosageregime of an alpha-4 agent is designed such that (1) the agentspecifically binds to alpha-4 integrin or an integrin, dimer comprisingalpha-4 integrin, and (2) the agent is administered repeatedly tomaintain alpha-4 integrin receptor saturation at a sufficient level tosuppress pathological inflammation. The agent of the invention can beuseful in the suppression of inflammation via binding and inhibition ofall integrin dimers comprising the alpha-4 subunit, or it can bedesigned to bind to a specific dimer, e.g., alpha-4 beta-1.

In one embodiment, the efficacy of a chronic dosage regime can bedetermined by the measurement of saturation of a specific integrindimer. In a particular example, it is believed that alpha-4 beta-1integrin dimer is involved in multiple sclerosis, and thus the level ofsaturation required for an efficacious chronic administration regime canbe measured via measurement of the saturation of the alpha-4 beta-1dimer receptor.

In another embodiment, where multiple integrin dimers are believed to beinvolved in a pathological inflammation, saturation of a combination ofdimer receptors can be measured to determine the efficacy of a chronicadministration regime. In a specific example, both the alpha-4 beta-1and the alpha-4 beta-7 dimers are believed to have involvement inpathological inflammation associated with inflammatory bowel disease.Thus, measurement of both alpha-4 beta-1 and alpha-4 beta-7 saturationlevels may be beneficial in determining the efficacy of a particularchronic administration regime.

In one embodiment, the success of a chronic dosage regime can bedetermined by assessing a physiological marker of the pathologicalinflammation. For example, in a chronic dosage regime administered forMS, the success of the dosage regime can be confirmed by detection ofthe levels of brain lesions using an imaging technique, e.g.; magneticresonance imaging (MRI). In another example, in a chronic dosage regimeadministered for CD, the success of the dosage regime can be confirmedby detection of the levels of serum C-reactive protein levels in apatient. In yet another example, the success of the dosage regime can bedetermined using a set group of criteria associated with wellness, e.g.,reduction in CDAI in a CD patient.

Another embodiment of the invention contemplates the treatment of aninflammatory disease of the gastrointestinal tract (e.g., Crohn'sdisease, ulcerative colitis and inflammatory bowel disease) of a subjectcomprising administering a therapeutically effective amount of acomposition comprising natalizumab sufficient to treat or amelioratesaid inflammatory disease of the gastrointestinal tract in said subject.

In a specific embodiment, the invention features a dosage regime whereinrepeated administration of an agent is provided to provide a level ofalpha-4 integrin receptor saturation of 65-100% in a patient, therebyproviding chronic suppression of pathological inflammation in thepatient. In another specific embodiment, the agent is repeatedlyadministered to provide levels of at least about 75-100% in a patient.In yet another specific embodiment, the agent in repeatedly administeredto provide levels of at least about 80-100% in a patient.

A feature of the invention is that undesirable effects of pathologicalinflammation can be suppressed long-term in a patient, e.g., over aperiod of six months, one year, two years, or more.

An aspect of the method of the invention is that sufficient levels of ananti-alpha-4 integrin agent are maintained over long periods to suppresspathological inflammation over those periods.

A feature of the invention is that the dosage form provides lower levelsof pathological inflammation over longer periods as compared to a singledose.

An advantage of the invention is that agents used in the methods of theinvention are well tolerated and have low toxicity.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the methods and formulations as more fully described below.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIG. 1 is a line graph illustrating the cumulative mean number of newGd-enhancing lesions in MS patients following dosing with natalizumab.

FIG. 2 shows the percentage of active scans at each time point duringthe natalizumab MS study. Active scans are those containing one or morenew Gd-enhancing lesions.

FIGS. 3A-C and 4A-C show the serum concentrations of natalizumabfollowing the dosage regime time points in an MS study. FIGS. 3A-C showthe levels for the 3-mg/kg study; FIGS. 4A-C show the levels for the6-mg/kg study.

FIGS. 5A-F illustrates the levels of receptor saturation maintained inthe MS study. Levels shown are percentage values.

FIGS. 6 and 7 show the percentage of Patients Achieving PredefinedCriteria of Clinical Response (FIG. 6) or Remission (FIG. 7) followingdosing in the CD study.

FIG. 8 illustrates mean changes in serum C-reactive protein in a subsetof patients who had elevated C-reactive protein at base-line in the CDstudy. Base-line values were (in mg/l): Placebo, 38.44 (N=26); 3+0 mg/kgGroup, 32.35 (N=38); 3+3 mg/kg Group, 41.16 (N=33); and 6+6 mg/kg Group,333 (N=26).

FIGS. 9 A-B demonstrate respectively the effects of natalizumab oncirculating eosinophils in patients with active Crohn's disease (CD) andulcerative colitis (UC). FIG. 9A demonstrates that natalizumabsignificantly increased circulating eosinophil counts in Crohn's disease(n=18) and ulcerative colitis patients (n=12) after 3 mg/kg natalizumabinfusion. FIG. 9B shows that administration of natalizumab significantlyincreased monocyte counts in active Crohn's disease and ulcerativecolitis patients after 3 mg/kg natalizumab infusion.

FIGS. 10 A-D demonstrate the impact of natalizumab administration onTCRαβ⁺ cells expressing activation antigen. Panel A demonstrates theeffect of natalizumab in patients with active Crohn's disease, i.e., asignificant increase of TCRαβ⁺ cells expressing CD26, HLA-DR, CD8CR andCD8 CD28 to at least four weeks post 3 mg/kg natalizumab infusion. PanelB demonstrates the effects of natalizumab on TCRαβ⁺ cells expressingactivation antigens in patients with activated ulcerative colitis, i.e.,significant increase in TCRαβ⁺ cells expressing CD26, HLA-DR, CD8DR andCD8CD28 to at least four weeks post 3 mg/kg natalizumab in fusion. PanelC demonstrates the effects of natalizumab administration on TCRαβ⁺ cellsexpressing activation antigens and memory and naïve markers in patientswith active Crohn's disease, i.e., significant increase of memory(CD45RO) and naïve (CD45RA) TCRαβ⁺ cells to at least four weekspost-natalizumab infusion. Panel B shows the effects of natalizumab onTCRαβ⁺ cells expressing activation antigens, memory and naïve markers inpatients with active ulcerative colitis, i.e., significant increase ofmemory (CD45RO), naïve (CD45RA), CD69 and CD38 TCRαβ⁺ cells at one weekpost natalizumab administration.

FIGS. 11 A-B demonstrate the effect of natalizumab on circulating TCRαβ⁺and NK-type cells in patients with active Crohn's disease and withulcerative colitis respectively.

DETAILED DESCRIPTION OF TILE INVENTION

Before the present methods and therapeutic agents are described, it isto be understood that this invention is not limited to particularmethods and therapeutic agents described, as such may, of course, vary.It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller, subject to any specifically excluded limit in the stated range.Where the stated range includes one or both of the limits, rangesexcluding either both of those included limits are also included in theinvention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein, the singular forms “a”, “and”, and“the” include plural referents unless the context clearly dictatesotherwise. Thus, for example, reference to “an antibody” includes aplurality of such antibodies and reference to “the dosage” includesreference to one or more dosages and equivalents thereof known to thoseskilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

Definitions

The term “anti-alpha-4 agent” as used herein refers to any agent, whichbinds specifically to an integrin comprising an alpha-4 subunit andinhibits activity of the integrin. This includes agents thatspecifically bind to alpha-4 integrin, as well as agents that bind to anintegrin dimer that comprises the alpha-4 integrin, e.g., alpha-4 beta-1(α4β1) or alpha-4 beta-7 (α4β7). The term “agents” is meant to includesynthetic and recombinant molecules (e.g., antibodies, small molecules,peptides, or other synthetically produced molecules or compounds, aswell as recombinantly produced gene products) as well asnaturally-occurring compounds (e.g., polypeptides, antibodies, and thelike).

The term “efficacy” as used herein in the context of a chronic dosageregime refers to the effectiveness of a particular treatment regime.Efficacy can be measured based on change the course of the disease inresponse to an agent of the present invention. For example, in thetreatment of MS, efficacy can be measured by the frequency of relapsesin relapsing-remitting MS, and by the presence or absence of new lesionsin the central nervous system as detected using methods such as MRI.

The term “success” as used herein in the context of chronic treatmentregimes refers to the effectiveness of a particular treatment regime.This includes a balance of efficacy, toxicity (e.g., side effects andpatient tolerance of a formulation or dosage unit), patient compliance,and the like. For a chronic administration regime to be considered“successful” it must balance different aspects of patient care andefficacy to produce the most favorable patient outcome.

The terms “specifically binds” or “binds specifically” as used hereinrefer to the situation in which one member of a specific binding pairwill not show any significant binding to molecules other than itsspecific binding partner (e.g., an affinity of about 1,000× or more forits binding partner). In the present invention, the anti-alpha-4integrin agent will not show significant binding to any polypeptideother than an alpha-4 integrin or a receptor comprising an alpha-4integrin. For example, antibodies used in the methods of the inventionthat bind to an alpha-4 integrin with a binding affinity of 10⁷ mole/1or more, preferably 10⁸ mole/liters or more, are said to bindspecifically to an alpha-4 integrin.

The term “substantially homologous” as used herein is intended to meanany polypeptide that has an alteration in the sequence such that afunctionally equivalent amino acid is substituted for one or more aminoacids in the polypeptide, thus producing a change that has no orrelatively little effect on the binding properties of the polypeptide.For example, one or more amino acid residues within the sequence can besubstituted by another amino acid of a similar polarity.

The terms “elicits an immune response” and “elicits a host immuneresponse” as used herein refer to the production of an immunologicalresponse to a receptor comprising an alpha-4 integrin in a subject uponintroduction of an agent of the invention to the subject. An immuneresponse in the subject can be characterized by serum reactivity with analpha-4 integrin receptor that is at least twice that of an untreatedsubject, more preferably three times the reactivity of an untreatedsubject, and even more preferably at least four times the reactivity ofan untreated subject, with serum immunoreactivity measured using a serumdilution of approximately 1:100.

The term “excipient material” is intended to mean any compound forming apart of the formulation that is intended to act merely as a carrier,i.e. not intended to have biological activity itself.

The term “adjuvant” as used herein refers to a composition additive thataugments the immune response to an agent of the invention but which willnot on its own elicit an immune response. Adjuvants may augment theimmune response using a variety of biological mechanisms, including hutnot limited to lymphocytic recruitment, T cell stimulation, B cellstimulation, and macrophage stimulation.

The terms “treating”, and “treatment” and the like are used herein togenerally mean obtaining a desired pharmacological and physiologicaleffect. The effect may be prophylactic in terms of preventing orpartially preventing a disease, symptom or condition thereof and/or maybe therapeutic in terms of a partial or complete cure of a disease,condition, symptom or adverse effect attributed to the disease. The term“treatment” as used herein covers any treatment of a disease in amammal, particularly a human, and includes: (a) preventing the diseasefrom occurring in a subject which may be predisposed to the disease buthas not yet been diagnosed as having it; (b) inhibiting the disease,i.e., arresting its development; or (c) relieving the disease, i.e.,causing regression of the disease and/or its symptoms or conditions. Theinvention is directed towards treating a patient's suffering fromdisease related to pathological inflammation. The present invention isinvolved in preventing, inhibiting, or relieving adverse effectsattributed to pathological inflammation over long periods of time and/orare such caused by the physiological responses to inappropriateinflammation present in a biological system over long periods of time.

The term “pathological inflammation” as used herein refers to aninappropriate and chronic inflammation associated with disordersincluding, but not limited to, asthma, atherosclerosis, AIDS dementia,diabetes, inflammatory bowel disease, rheumatoid arthritis, transplantrejection, graft versus host disease, multiple sclerosis (especially toinhibit further demyelination), tumor metastasis, nephritis, atopicdermatitis, psoriasis, myocardial ischemic, chronic prostatitis,complications from sickle cell anemia, lupus erythematosus, and acuteleukocyte mediated lung injury. Such inflammation is characterized by aheightened response of inflammatory cells, including infiltratingleukocytes. Over time, such pathological inflammation often results indamage to tissue in the region of inappropriate inflammation.

By “Antegren®” is meant to include the antibody also known as AN100226(antibody code number) or natalizumab (USAN name). Antegren® is arecombinant, humanized anti-alpha-4 integrin antibody. Preferably thedisease or condition being treated in the mammal is one which ismodulated when a therapeutically effective dose of Antegren® isadministered.

General Aspects of the Invention

The present invention is based on the surprising result that chronicadministration of an emerging class of new compounds known as selectiveadhesion molecule inhibitors (SAMIs) is sufficient to provide themaintenance of chronic suppression of inflammation in disordersinvolving integrin dimers. Upon cessation of the repeated dosage regime,the suppression of the inflammation is reversed (see, e.g., FIG. 2).Previous treatments of inflammatory inhibitors have approached thedosage regimes quite differently, in the belief that the administrationof an inflammatory inhibitor would cause a reaction of the body's ownresponse system, which would in turn lead to a recognition of theinflammation as pathological and a resulting chronic relief of thepathological inflammation. What the inventors have shown herein is thata chronic dosage regime is not only more effective than a short-termdosage regime, but in fact it is required to maintain the suppression ofpathological inflammation. Thus, in order to realize some of the moreimportant advantages of the invention, the levels of an anti-alpha-4integrin agent need to be maintained over a number of months or evenyears.

The present invention is based on the results of a large, randomized,placebo-controlled trial of an anti-alpha-4 integrin antibody,natalizumab, in patients with relapsing MS or with moderate to severelyactive CD. Natalizumab is a recombinant, humanized, monoclonal antibodyantagonist against alpha-4 integrin. Results from these two trials haveshown that treatment with natalizumab improved the signs and symptoms ofpatients with MS and CD. The invention is also intended to include otherchimeric antibodies, including Primatized™ antibodies.

In a general sense the method of the invention does not involve anyparticular mode of administration, since the mode of administration isdependent upon the form of the active agent and the formulationdeveloped to administer the active agent(s). However, the specificexamples described here were obtained using parenteral administration ofnatalizumab. Although the present invention is described using anantibody that specifically binds to alpha-4 integrin, it is alsointended to include chronic administration of, for example, bivalent ormultivalent antibodies that recognize both partners of an integrindimer, provided the dimer comprises an alpha-4 integrin.

The general concept of the invention relates to introducing relativelyconstant amounts of an active agent to a patient's circulatory systemover a period of months or years. This chronic introduction of an agentthat selectively binds to alpha-4 integrin or a dimer comprising alpha-4integrin results in suppression of pathological inflammation beingmaintained at a constant level over a period of time. By maintainingtherapeutic levels of an active agent for a period of time, pathologicalinflammation can be chronically suppressed in the patient.

In a very specific sense, the invention involves obtaining andmaintaining a receptor saturation level in a human patient of a dimercomprising alpha-4 integrin in a range of from about 65% to about 100%,more preferably between about 75% to about 100%, and even morepreferably between about 80% to about 100%. These receptor saturationlevels are maintained at these levels chronically (e.g., over a periodof 6 months or so) to allow for continued suppression of pathologicalinflammation.

Agents that Selectively Bind to Alpha-4 Integrins

Various types of agents with the ability to bind to and inhibit alpha-4integrin activity can be used in the practice of the invention. Manysuch agents have been identified and characterized, and specific agentsare described below. Given the teachings disclosed herein, it is wellwithin the skill of one in the art to identify other agents that will beable to inhibit the alpha-4-comprising integrin dimers in a manner thatbiologically mimics or is similar to the specifically described agents,and the present invention is intended to include the chronicadministration of such agents and combinations of such agents.

Antibodies

In one specific embodiment, the agents of the invention are antibodiesor immunologically active fragments thereof that selectively bind to analpha-4 integrin or a dimer comprising alpha-4, such as alpha-4 beta-1or alpha-4 beta-7.

When the agent of the invention is an antibody, a monoclonal antibody ispreferred. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentepitopes, each monoclonal antibody is directed against a single epitopeon the antigen. A second advantage of monoclonal antibodies is that theyare synthesized by means that are uncontaminated by otherimmunoglobulins, e.g., by phage display or isolation from a hybridoma.Although the present invention intends to encompass both polyclonal andmonoclonal antibodies as agents of the invention, monoclonal antibodiesare preferred as they are highly specific, and the invention is thusdiscussed primarily in terms of monoclonal antibodies.

In addition, other antibodies can be identified using techniquesavailable in the art. For example, monoclonal antibodies of the presentinvention can be produced using phage display technology. Antibodyfragments that selectively bind to an alpha-4 integrin or a dimercomprising an alpha-4 integrin are then isolated. Exemplary preferredmethods for producing such antibodies via phage display are disclosed inU.S. Pat. Nos. 6,225,447; 6,180,336; 6,172,197; 6,140,471; 5,969,108;5,885,793; 5,872,215; 5,871,907; 5,858,657; 5,837,242; 5,733,743; and5,565,332.

Monoclonal antibodies can also be produced using the conventionalhybridoma methods. These methods have been widely applied to producehybrid cell lines that secrete high levels of monoclonal antibodiesagainst many specific antigens, and can also be used to producemonoclonal antibodies of the present invention. For example, mice (e.g.,Balb/c mice) can be immunized with an antigenic alpha-4 integrin epitopeby intraperitoneal injection. After sufficient time has passed to allowfor an immune response, the mice are sacrificed, and the spleen cellsobtained and fused with myeloma cells, using techniques well known inthe art. The resulting fused cells, hybridomas, are then grown in aselective medium, and the surviving cells grown in such medium usinglimiting dilution conditions. After cloning and recloning, hybridomascan be isolated that secrete antibodies (for example, of the IgG or IgMclass or IgG1 subclass) that selectively bind to the target, alpha-4integrin or a dimer comprising an alpha-4 integrin. To produce agentsspecific for human use, the isolated monoclonal can then be used toproduce chimeric and humanized antibodies.

Antibodies of the invention include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized or chimeric antibodies,single chain antibodies (e.g., scFv), Fab fragments, F(ab′) fragments,fragments produced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of theinvention), and epitope-binding fragments of any of the above. Mostpreferably the antibodies are human antigen-binding antibody fragmentsof the present invention and include, but are not limited to, Fab, Fab′and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VHdomain. Antigen-binding antibody fragments, including single-chainantibodies, may comprise the variable region(s) alone or in combinationwith the entirety or a portion of the following: hinge region, CH1, CH2,and CH3 domains. Also included in the invention are antigen-bindingfragments that can comprise any combination of variable region(s) with ahinge region, CH1, CH2, and CH3 domains. The antibodies of the inventionmay be from any animal origin including birds and mammals. Preferably,the antibodies are human, murine (e.g., mouse and rat), donkey, sheep,monkey, rabbit, goat, guinea pig, pig, camel, horse, or chicken (orother avian). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described infra and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al.

Chimeric and humanized antibodies can be produced from non-humanantibodies, and can have the same or similar binding affinity as theantibody from which they are produced. Techniques for producing chimericantibodies (Morrison et al., 1984 Proc. Natl. Acad. Sci. 81: 6851;Neuberger et al., 1984 Nature 312: 604; Takeda et al., 1985 Nature 314:452) include splicing the genes from, e.g., a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity; such antibodiesare within the scope of this invention. For example, a nucleic acidencoding a variable (V) region of a mouse monoclonal antibody can bejoined to a nucleic acid encoding a human constant (C) region, e.g.,IgG1 or IgG4. The resulting antibody is thus a species hybrid, generallywith the antigen binding domain from the non-human antibody and the C oreffector domain from a human or primate antibody.

Humanized antibodies are antibodies with variable regions that areprimarily from a human antibody (i.e., the acceptor antibody), but whichhave complementarity determining regions substantially from a non-humanantibody (the donor antibody). See, e.g., Queen et al., Proc. Nat'l.Acad. Sci USA 86: 10029-10033 (1989); WO 90/07861, U.S. Pat. Nos.6,054,297; 5,693,761; 5,585,089; 5,530,101; and 5,224,539. The constantregion or regions of these antibodies are generally also from a humanantibody. The human variable domains are typically chosen from humanantibodies having sequences displaying a high homology with the desirednon-human variable region binding domains. The heavy and light chainvariable residues can be derived from the same antibody, or a differenthuman antibody. In addition, the sequences can be chosen as a consensusof several human antibodies, such as described in WO 92/22653.

A “Primatized™ antibody” is a recombinant antibody containing primatevariable sequences or antigen binding portions, and human constantdomain sequences. See Newman, Biotechnology, 1992, 10: 1455-60.Primatization of antibodies results in the generation of antibodies thatcontain monkey variable domains and human constant sequences. For moredetails see U.S. Pat. No. 6,113,898. This technique modifies antibodiessuch that they are not rejected upon administration in humans becausethey are antigenic. This technique relies on immunization of cynomolgusmonkeys with human antigens or receptors. This technique was developedto create high affinity monoclonal antibodies directed to human cellsurface antigens.

Specific amino acids within the human variable region are selected forsubstitution based on the predicted conformation and antigen bindingproperties. This can be determined using techniques such as computermodeling, prediction of the behavior and binding properties of aminoacids at certain locations within the variable region, and observationof effects of substitution. For example, when an amino acid differsbetween a non-human variable region and a human variable region, thehuman variable region can be altered to reflect the amino acidcomposition of the non-human variable region.

In a specific embodiment, the antibodies used in the chronic dosageregime of the present invention are humanized antibodies as disclosed inU.S. Pat. No. 5,840,299, which is incorporated herein by reference.

In another embodiment, transgenic mice containing human antibody genescan be immunized with an antigenic alpha-4 integrin structure andhybridoma technology can be used to generate human antibodies thatselectively bind to alpha-4 integrin.

Chimeric, human and/or humanized antibodies can be produced by usingrecombinant expression, e.g., expression in human hybridomas (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985)), in myeloma cells or in Chinese hamster ovary (CHO) cells.Alternatively, antibody coding sequences can be incorporated intotransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal. See e.g.,U.S. Pat. Nos. 6,197,946; 5,849,992; 5,565,362; 5,336,894; and5,304,489. Suitable transgenes include transgenes having a promoterand/or enhancer from a mammary gland specific gene, for example caseinor β-lactoglobulin.

Small Molecules

Small molecules for use in the present invention encompass numerouschemical classes, though typically they are organic molecules,preferably small organic compounds having a molecular weight of morethan 50 and less than about 4,000 Daltons. Candidate agents comprisefunctional groups necessary for structural interaction with proteins,particularly hydrogen bonding, and typically include at least an amine,carbonyl, hydroxyl or carboxyl group, preferably at least two offunctional chemical groups. The candidate agents often comprise cyclicalcarbon or heterocyclic structures and/or aromatic or polyaromaticstructures substituted with one or more of the above functional groups.Candidate agents are also found among biomolecules including, but notlimited to: peptides, saccharides, fatty acids, steroids, purines,pyrimidines, derivatives, structural analogs or combinations thereof.

The small molecules can be obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs.

Anti-Alpha-4 Integrin Peptides

The methods of the invention can be performed with any peptide that iscapable of binding to an alpha-4 integrin or a dimer comprising analpha-4 subunit. Included in the methods of the invention are peptidesthat are substantially homologous to a region of the extracellularmatrix or a natural ligand of the specific alpha-4 integrin receptor orreceptors targeted. For example, for the chronic inhibition of alpha-4beta-1 receptor, peptides can be used that comprise at least a portionof the fibronectin IIICS region (e.g., peptides comprising at least aportion of the CS-1 peptide sequence or a sequence substantiallyhomologous to the CS-1 sequence) can be used to bind to a receptor andinhibit the activity of the alpha-4 comprising integrin. See for exampleU.S. Ser. No. 08/452,098, which is incorporated by reference in itsentirety.

Agents that Elicit an Immune Response

In a specific embodiment, the agents of the invention are peptides orpeptidomimetics that comprise an immunogenic fragment of an alpha-4integrin. An immunogenic fragment is any fragment that comprises anepitope of alpha-4, and generally has at least 3, 5, 7, 10, 15, 17 or 20contiguous amino acids from a naturally occurring mammalian alpha-4protein. The peptide sequence of both human and murine alpha-4 isaccessible from GenBank (Accession Nos. AA59613 and NP_034706). Oneskilled in the art can readily design a peptide agent of the inventionbased on the amino acid sequence of alpha-4 or using the wild-typenucleotides that encode them (e.g., GenBank Accession Nos. L12002 andNM_01056, respectively). Once an appropriate peptide is designed, it canbe initially screened against antibodies known to have the desiredimmunogenic activity, e.g., antibodies that selectively bind to analpha-4 structure and are characterized by the ability to inhibit theactivity of an integrin comprising alpha-4.

The immunogenic fragment may also be designed to have amino acid analogsor other structural elements that will enhance the immunogenic response.In particular, peptide fragments may have altered C- or N-terminal endsthat enhance overall immunogenicity of the molecules while not impedingtheir ability to elicit an immune response. Examples of such analogsinclude, but are not limited to: alpha, alpha-disubstituted amino acids,N-alkyl amino acids, lactic acid, 4-hydroxyproline,gamma-carboxyglutamate, gamma-N,N,N,-trimethyllysine,gamma-N-acetyllysine, O-phosphoserine, N-acetylserine,N-formylmethionine, 3-methylhistidine, and 5-hydroxylysine. Other usefulanalogs can be found in Sigma, Biochemicals and Reagents, Sigma-Aldrich(2001). The fragment may also be detectably labeled to allow fortracking of the molecule within a subject following administration to asubject.

Peptides, analog structures, peptidomimetics and the like can beisolated from natural sources, and then optionally processed (e.g., viapeptide cleavage) or alternatively synthesized by conventionaltechniques known in the art such as solid phase synthesis or recombinantexpression. See, e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual (Cold Spring Harbor Press, New York, 2^(nd) edition 1989).Automatic peptide synthesis can be performed using a commerciallyavailable apparatus from manufacturers such as Applied Biosystems(Foster City, Calif.), and methods of doing so are well established.Recombinant production of the proteins may be in prokaryotic, such asphage or bacterial cells or eukaryotic systems, such as yeast, insect,or mammalian cells. Alternatively, proteins can be produced usingcell-free in vitro systems known in the art.

In another example, phage peptide display libraries can be used toexpress large numbers of peptides that can be screened in vitro toidentify peptides that specifically bind to alpha-4 or a dimercomprising an alpha-4 integrin. Phage display technology provides ameans for expressing a diverse population of random or selectivelyrandomized peptides. Various methods of phage display and methods forproducing diverse populations of peptides are well known in the art. Forexample, Ladner et al. (U.S. Pat. No. 5,223,409), describes methods forpreparing diverse populations of binding domains on the surface of aphage. Ladner et al. describe phage vectors useful for producing a phagedisplay library, as well as methods for selecting potential bindingdomains and producing randomly or selectively mutated binding domains.Screening of a phage display library generally involves in vitro palmingof the library using a purified target molecule. Phage that bind thetarget molecule can be recovered; individual phage can be cloned, andthe peptide expressed by a cloned phage can be determined.

Similarly, Smith and Scott (Meth. Enzymol. 217: 228-257 (1993) andScience 249: 386-390 (1990)) describe methods of producing phage peptidedisplay libraries, including vectors and methods of diversifying thepopulation of peptides that are expressed (see, also, Huse, WO 91/07141and WO 91/07149). Phage display technology can be particularly powerfulwhen used, for example, with a codon based mutagenesis method, which canbe used to produce random peptides or desirably biased peptides (see,e.g., U.S. Pat. No. 5,264,563). These and other well known methods canbe used to produce a phage display library, which can be subjected tothe in vivo panning method of the invention in order to identify apeptide that homes to one or a few selected organs.

The molecules of a peptide phage display library also can be present asa conjugate, which can facilitate recovery or identification of thepeptide of interest. As used herein, the term “conjugate” means apeptide or peptidomimetic of the library linked to a physical, chemicalor biological moiety such as but not limited to a solid substrate, aplastic microbead, an oligonucleotide or a bacteriophage, and the like.The moiety can provide a means to identify or recover an agent.

Some agents used to elicit an immune response mimic the appropriateepitope for inducing an immune response against alpha-4 integrin but aretoo small to be immunogenic on their own. In this situation, a peptideagent can be linked to a suitable carrier to facilitate an immuneresponse. Suitable carriers include serum albumins, keyhole limpethemocyanin (KLH), immunoglobulin molecules, thyroglobulin, ovalbumin,tetanus toxoid, or a toxoid from other pathogenic bacteria, such asdiphtheria, E. coli, cholera, or H. pylori, or an attenuated toxinderivative. Other carriers include T-cell epitopes that bind to multipleMHC alleles, e.g., at least 75% of all human MHC alleles. Such carriersare sometimes known in the art as “universal T-cell epitopes.” Examplesof universal T-cell epitopes include:

Influenza Hemagluttinin: PKYVKQNTLKLAT HA₃₀₇₋₃₁₉ (SEQ ID NO: 1) PADREAKXVAAWTLKAAA (SEQ ID NO: 2), where X is preferably cyclohexylalanine,tyrosine, or phenylalanine Malaria CS: T3 epitope EKKIAKMEKASSVFNV(SEQ ID NO: 3) Hepatitis B surface  FFLLTRILTI antigen: HBsAg₁₉₋₂₈(SEQ ID NO: 4) Heat Shock Protein 65: DQSIGDLIAEAMDKVGNEG hsp65₁₅₃₋₁₇₁(SEQ ID NO: 5) bacille Calmette-Guerin QVHFQPLPPAVVKL (SEQ ID NO: 6)Tetanus toxoid: QYIKANSKFIGITEL TT₈₃₀₋₈₄₄ (SEQ ID NO: 7) Tetanus toxoid:FNNFTVSFWLRVPKVSASHLE TT₉₄₇₋₉₆₇ (SEQ ID NO: 8) HIV gp120 T1KQIINMWQEVGKAMYA. (SEQ ID NO: 9)

Other carriers for stimulating or enhancing an immune response includecytokines such as IL-1, IL-1 α and β peptides, IL-2, γ-INF, IL-10,GM-CSF, and chemokines, such as macrophage inflammatory protein (MIP)1αand β and RANTES (i.e., regulation upon activation normal T cellexpressed and secreted). Immunogenic agents can also be linked topeptides that enhance transport across tissues, as described in WO97/17613 and WO 97/17614.

Immunogenic agents can be linked to carriers by chemical cross-linking.Techniques for linking an agent to a carrier include but are not limitedto the formation of disulfide linkages usingN-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and succinimidyl4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) (if the peptidelacks a sulfhydryl group, this can be provided by addition of a cysteineresidue). These reagents create a disulfide linkage between themselvesand a peptide cysteine that resides on one protein and an amide linkagethrough the ε-amino on a lysine, or other free amino group in otheramino acids. A variety of such disulfide/amide-forming agents aredescribed in Immun. Rev. 62: 185 (1982). Other bifunctional couplingagents form a thioether rather than a disulfide linkage. Many of thesethio-ether-forming agents are commercially available and includereactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid,2-iodoacetic acid, and 4-(N-maleimido-methyl)cyclohexane-1-carboxylicacid. The carboxyl groups can be activated by combining them withsuccinimide or 1-hydroxyl-2-nitro-4-sulfonic acid sodium salt.

Peptide agents can also be expressed as fusion proteins with carriers(i.e., heterologous peptides). The peptide agent can be linked at itsamino terminus, its carboxyl terminus, or both to a carrier. Optionally,multiple repeats of the immunogenic peptide can be present in the fusionprotein. Optionally, an immunogenic peptide can be linked to multiplecopies of a heterologous peptide, for example, at both the N- andC-termini of the peptide. Some carrier peptides serve to induce a helperT-cell response against the carrier peptide. The induced helper T-cellsin turn induce a B-cell response against the immunogenic peptide linkedto the carrier peptide.

Whether the agent is an antibody, polypeptide, peptide, small moleculeor other pharmaceutically useful compound according to the presentinvention that is to be given to an individual, administration is via achronic dosage regime. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g., decisions ondosage etc., is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.Examples of the techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 18th edition, Osol, A. (ed.), 1990.

Pharmaceutical Compositions

The present invention also provides pharmaceutical compositions for thereduction of chronic pathological inflammation in a subject susceptibleto such and/or suffering from a disorder associated with pathologicalinflammation.

Pharmaceutical formulations of the invention preferably contain an agentin a concentration from about 0.1 to about 10% of the formulation. Theymay also be used in appropriate association with other pharmaceuticallyactive compounds. The following methods and excipients are merelyexemplary and are in no way meant to be limiting.

For oral preparations, the agents can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

When the agent is an antibody, the formulation is preferablyadministered in a parenteral dosage form. The preferred form depends onthe intended mode of administration and therapeutic application. Thecompositions can also include, depending on the formulation desired,pharmaceutically acceptable, non-toxic carriers or diluents, which aredefined as vehicles commonly used to formulate pharmaceuticalcompositions for animal or human administration. The diluent is selectedso as not to affect the biological activity of the combination. Examplesof such diluents are distilled water, physiological phosphate-bufferedsaline, Ringer's solutions, dextrose solution, and Hank's solution. Inaddition, the pharmaceutical composition or formulation may also includeother carriers, adjuvants, or nontoxic, nontherapeutic, non-immunogenicstabilizers and the like. Also included may be carrier molecules such asproteoglycans. Specific examples of such carrier molecules include, butare not limited to, glycosaminoglycans such as heparin sulfate,hyaluronic acid, keratan-sulfate, chondroitin 4-sulfate, chondroitin6-sulfate, heparan sulfate and dermatin sulfate, perlecan, andpentopolysulfate.

Antibodies of the invention can be administered as injectionable dosagesof a solution or as a suspension of the substance in a physiologicallyacceptable diluent with a pharmaceutical carrier that can be a sterileliquid such as water and oils with or without the addition of asurfactant. Other pharmaceutically preparations are those of petroleum,animal, vegetable, or synthetic origin, for example, peanut oil, soybeanoil, and mineral oil. In general, glycols such as propylene glycol orpolyethylene glycol are preferred liquid carriers, particularly forinjectable solutions. The agents of this invention can be administeredin a sustained release form, for example a depot injection, implantpreparation, or osmotic pump, which can be formulated in such a manneras to permit a sustained release of the active ingredient.

In addition, agents of the invention that are antibodies may be providedby administering a polynucleotide encoding a whole or partial antibody(e.g., a single chain Fv) to a subject. The polynucleotide isadministered to a subject in an appropriate vehicle to allow theexpression of the antibody in the subject in a therapeutically effectiveamount.

The agents of the invention can be formulated into preparations forinjections by dissolving, suspending or emulsifying them in an aqueousor nonaqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol. The formulations may also contain conventionaladditives such as solubilizers, isotonic agents, suspending agents,emulsifying agents, stabilizers and preservatives.

The agents can be utilized in aerosol formulation to be administered viainhalation or pulmonary delivery. The agents of the present inventioncan be formulated into pressurized acceptable propellants such asdichlorodifluoromethane, propane, nitrogen and the like.

Furthermore, the agents can be made into suppositories by mixing with avariety of bases such as emulsifying bases or water-soluble bases. Theagents of the present invention can be administered rectally via asuppository. The suppository can include vehicles such as cocoa butter,carbowaxes and polyethylene glycols, which melt at body temperature, yetare solidified at room temperature.

Administration of an agent of the invention may be accomplished by anyconvenient means, including parenteral injection, and may be systemic orlocalized in delivery. The agents of this invention can be incorporatedinto a variety of formulations for therapeutic administration. Moreparticularly, the agents of the present invention can be formulated intopharmaceutical compositions by combination with appropriatepharmaceutically acceptable carriers or diluents, and may be formulatedinto preparations in solid, semi-solid, liquid or gaseous forms, such astablets, capsules, powders, granules, ointments, solutions,suppositories, injections, inhalants, gels, microspheres, and aerosols.As such, administration of the agents can be achieved in various ways,including oral, buccal, rectal, parenteral, intraperitoneal,intradermal, transdermal, intratracheal, intranasal, gastric,intramuscular, intracranial, subdermal etc., administration. The activeagent may be systemic after administration or may be localized by theuse of regional administration, intramural administration, or use of animplant that acts to retain the active dose at the site of implantation.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more agents ofthe present invention. Similarly, unit dosage forms for injection orintravenous administration may comprise the agent of the presentinvention in a composition as a solution in sterile water, normal salineor another pharmaceutically acceptable carrier.

Implants for sustained release formulations are well known in the art.Implants are formulated as microspheres, slabs, etc. with biodegradableor non-biodegradable polymers. For example, polymers of lactic acidand/or glycolic acid form an erodible polymer that is well tolerated bythe host. The implant is placed in proximity to the site of proteindeposits (e.g., the site of formation of amyloid deposits associatedwith neurodegenerative disorders), so that the local concentration ofactive agent is increased at that site relative to the rest of the body.

A typical dosage unit for administration of a subject includes, but isnot limited to: a solution suitable for intravenous administration; atablet taken from two to six times daily; or a one time-release capsuleor tablet taken once a day and containing a proportionally highercontent of active ingredient, etc. The time-release effect may beobtained by capsule materials that dissolve at different pH values, bycapsules that release slowly by osmotic pressure or by any other knownmeans of controlled release.

Certain agents of the invention, including antibodies and peptides, aresometimes administered in combination with an adjuvant. A variety ofadjuvants can be used in combination with an anti alpha-4 integrin agentto elicit an immune response. Preferred adjuvants augment the intrinsicresponse to an agent without causing conformational changes in the agentthat affect the qualitative form of the response. Preferred adjuvantsinclude aluminum hydroxide and aluminum phosphate, 3 De-O-acylatedmonophosphoryl lipid A (MPL™) (see GB 2220211 (RIBI ImmunoChem ResearchInc., Hamilton, Mont., now part of Corixa). Stimulon™ QS-21 is atriterpene glycoside or saponin isolated from the bark of the QuillajaSaponaria Molina tree found in South America (see Kensil et al., inVaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman,Plenum Press, NY, 1995); U.S. Pat. No. 5,057,540, (AquilaBioPharmaceuticals, Framingham, Mass.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., 1997 N. Engl. J. Med. 336: 86-91). Another adjuvant is CpG (WO98/40100). Alternatively, an agent can be coupled to an adjuvant.However, such coupling should not substantially change the conformationof the desired alpha-4 epitope so as to affect the nature of the hostimmune response. Adjuvants can be administered as a component of atherapeutic composition with an active agent or can be administeredseparately, before, concurrently with, or after administration of thetherapeutic agent.

A preferred class of adjuvants for administration is aluminum salts(alum), such as aluminum hydroxide, aluminum phosphate, and aluminumsulfate. Such adjuvants can be used with or without other specificimmunostimulating agents such as MPL or 3-DMP, QS-21, polymeric ormonomeric amino acids such as polyglutamic acid or polylysine. Anotherclass of adjuvants is oil-in-water emulsion formulations. Such adjuvantscan be used with or without other specific immunostimulating agents suchas muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine(thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE),N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™, or other bacterial cell wallcomponents. Oil-in-water emulsions include (a) MF59 (WO 90/14837),containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionallycontaining various amounts of MTP-PE) formulated into submicronparticles using a microfluidizer such as Model 110Y microfluidizer(Microfluidics, Newton Mass.), (b) SAF, containing 10% Squalene, 0.4%Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphoryl lipid A, trehalose dimycolate (TDM), andcell wall skeleton (CWS), preferably MPL+CWS (Detox™). Another class ofpreferred adjuvants is saponin adjuvants, such as Stimulon™ (QS-21;Aquila, Framingham, Mass.) or particles generated therefrom, such asISCOMs (immunostimulating complexes) and ISCOMATRIX. Other adjuvantsinclude Complete and Incomplete Freund's Adjuvant (IFA), cytokines, suchas interleukins (IL-1, IL-2, and IL-12), macrophage colony stimulatingfactor (M-CSF), and tumor necrosis factor (TNF). Such adjuvants aregenerally available from commercial sources.

An adjuvant can be administered with an agent as a single composition,or can be administered before, concurrent with or after administrationof the agent. The agent and an adjuvant can be packaged and supplied inthe same vial or can be packaged in separate vials and mixed before use.The agent and adjuvant are typically packaged with a label indicatingthe intended therapeutic application. If the agent and adjuvant arepackaged separately, the packaging typically includes instructions formixing before use. The choice of an adjuvant and/or carrier depends onsuch factors as the stability of the formulation containing theadjuvant, the route of administration, the dosing schedule, and theefficacy of the adjuvant for the species being vaccinated. In humans, apreferred pharmaceutically acceptable adjuvant is one that has beenapproved for human administration by pertinent regulatory bodies.Examples of such preferred adjuvants for humans include alum, MPL andQS-21. Optionally, two or more different adjuvants can be usedsimultaneously. Preferred combinations include alum with MPL, alum withQS-21, MPL with QS-21, and alum, QS-21 and MPL together. Also,Incomplete Freund's adjuvant can be used (Chang et al., 1998 AdvancedDrug Delivery Reviews 32: 173-186), optionally in combination with anyof alum, QS-21, and MPL and all combinations thereof.

Chronic Administration Dosage Regimes

The chronic treatment regimes of the present invention providesanti-alpha-4 integrin agent at a level that will maintain sufficientreceptor saturation to suppress pathological inflammation in a patientin need of such. The methods of the invention entail administration onceper every two weeks or once a month to once every two months, withrepeated dosings taking place over a period of at least six months, andmore preferably for a year or longer. The methods of the inventioninvolve obtaining and maintaining a receptor saturation level in a humanpatient of a dimer comprising alpha-4 integrin (e.g., VLA-4) in a rangeof from about 65% to 100%, more preferably between about 75% to about100%, and even more preferably between about 80% to about 100%. Thesereceptor saturation levels are maintained at these levels chronically(e.g., over a period of 6 months or so) to allow for continuedsuppression of pathological inflammation.

In a specific embodiment, the anti-alpha-4 agent is an antibody,preferably a humanized or human antibody (e.g., natalizumab), and thedosing is on a monthly basis. Levels of receptor saturation can bemonitored to determine the efficacy of the dosing regime, andphysiological markers measured to confirm the success of the dosageregime. As a confirmation, serum levels of the antibody can be monitoredto identify clearance of the antibody and to determine the potentialeffect of half-life on the efficacy of the treatment.

The amount of agent administered in a dosage unit may depend on whetheradjuvant is also administered, with higher dosages generally beingrequired in the presence of adjuvant. For immunization with an agent ofthe invention, the dosage ranges from about 0.0001 to about 100 mg/kg,and more usually about 0.01 to 5 mg/kg, of the host body weight. Forexample, dosages can be about 1 mg/kg body weight or about 10 mg/kg bodyweight. Dosage and frequency may vary depending on the half-life of theagent in the patient. The dosage and frequency of administration canvary depending on whether the treatment is prophylactic or therapeutic.For antibody administration, each dosing injection is generally betweenabout 2.0 to about 8.0 mg/kg. In accordance with the teachings providedherein, effective dosages can be monitored by obtaining a fluid samplefrom the patient, generally a blood serum or cerebrospinal fluid sample,and determining the integrin receptor saturation using methods wellknown in the art. Ideally, a sample is taken prior to initial dosing;subsequent samples are taken and measured prior to and/or after eachimmunization. A particularly preferred amount is a 3 mg per kg ofpatient per month of natalizumab or an immunologically active fragmentequivalent thereof.

When adjuvant is being administered, the dosage level is increased inaccordance with the particular adjuvant and the level of immunogenicityof the anti-alpha-4 agent. Doses for individual agents, selected inaccordance with the present invention, are determined according tostandard dosing methods, taken in conjunction with the teachingsprovided herein.

As an alternative to chronic administration comprised of repeatedindividual dosings, an anti-alpha-4 agent can be administered as asustained release formulation, provided the dosage is such that thelevels of receptor saturation remain sufficient to suppressinflammation. For example, controlled release systems can be used tochronically administer an anti-alpha-4 agent within the scope of thisinvention. Discussions of appropriate controlled release dosage formsmay be found in Lesczek Krowczynski, Extended-Release Dosage Forms, 1987(CRC Press, Inc.).

The various controlled release technologies cover a very broad spectrumof drug dosage forms. Controlled release technologies include, but arenot limited to physical systems and chemical systems. Physical systemsinclude, but are not limited to, reservoir systems with rate-controllingmembranes, such as microencapsulation, macroencapsulation, and membranesystems; reservoir systems without rate-controlling membranes, such ashollow fibers, ultra microporous cellulose triacetate, and porouspolymeric substrates and foams; monolithic systems, including thosesystems physically dissolved in non-porous, polymeric, or elastomericmatrices (e.g., non-erodible, erodible, environmental agent ingression,and degradable), and materials physically dispersed in non-porous,polymeric, or elastomeric matrices (e.g., non-erodible, erodible,environmental agent ingression, and degradable); laminated structures,including reservoir layers chemically similar or dissimilar to outercontrol layers; and other physical methods, such as osmotic pumps, oradsorption onto ion-exchange resins.

Chemical systems include, but are not limited to, chemical erosion ofpolymer matrices (e.g., heterogeneous, or homogeneous erosion), orbiological erosion of a polymer matrix (e.g., heterogeneous, orhomogeneous). Additional discussion of categories of systems forcontrolled release may be found in Agis F. Kydonieus, Controlled ReleaseTechnologies: Methods, Theory and Applications, 1980 (CRC Press, Inc.).

The methods of the invention can be used to treat a patient that isaffected with a disorder involving or arising from pathologicalinflammation, or to prophylactically treat a patient at risk for aparticular disorder. The dosage regimes that are necessary forprophylactic versus therapeutic treatment can vary, and will need to bedesigned for the specific use and disorder treated.

In some methods, two or more agents (e.g., monoclonal antibodies withdifferent binding specificities) are administered simultaneously, inwhich case the dosage of each agent administered falls within the rangesindicated. Intervals can also be irregular as indicated by measuringreceptor saturation levels or by following other indicia of the diseaseprocess.

Those of skill will readily appreciate that dose levels can vary as afunction of the specific agent, the severity of the symptoms and thesusceptibility of the subject to side effects. Some of the specificagents are more potent than others. Preferred dosages for a given agentare readily determinable by those of skill in the art by a variety ofmeans. A preferred means is to measure the physiological potency of agiven agent.

Therapeutic Indications

The controlled release formulations of the present invention can be usedto obtain a wide range of desirable effects. Particularly theformulations of the invention are useful in treating essentially anydisease state or symptom that is treatable by long term administrationof anti-inflammatories that target pathological inflammation.

The invention also provides methods of treatment that exploit theability of anti-alpha-4 integrin agents to block alpha-4-dependentinteractions. The alpha-4-dependent interaction with the VCAM-1 ligandon endothelial cells is an early event in many inflammatory responses,including those of the central nervous system. Undesired diseases andconditions resulting from inflammation and having acute and/or chronicclinical exacerbations include multiple sclerosis (Yednock et al., 1992Nature 356: 63; Baron et al., 1993. J. Exp. Med. 177: 57), meningitis,encephalitis, stroke, other cerebral traumas, inflammatory bowel disease(IBD) including ulcerative colitis and Crohn's disease (CD) (Hamann etal., 1994 J. Immunol. 152: 3238), (Podolsky et al., 1993 J. Clin.Invest. 92: 372), rheumatoid arthritis (van Dinther-Janssen et al., 1991J. Immunol. 147: 4207; van Dinther-Janssen et al., 1993 Annals RheumaticDiseases 52: 672); Elices et al., 1994 J. Clin. Invest. 93: 405);Postigo et al., 1992 J. Clin. Invest. 89: 1445), asthma (Mulligan etal., 1993 J. Immunol. 150: 2407) and acute juvenile onset diabetes(Type 1) (Yang et al., 1993 PNAS 90: 10494); Burkly et al., 1994Diabetes 43: 529); Baron et al., 1994 J. Clin. Invest. 93: 1700), AIDSdementia (Sasseville et al., 1994 Am. J. Path. 144: 27); atherosclerosis(Cybulsky et al., 1991 Science 251: 788-91, Li et al., 1993Arterioscler. Thromb. 13: 197), nephritis (Rabb et al., 1995 SpringerSemin. Immunopathol. 16: 417-25), retinitis, atopic dermatitis,psoriasis, myocardial ischemia, chronic prostatitis, complications fromsickle cell anemia, lupus erythematosus, and acute leukocyte-mediatedlung injury such as occurs in adult respiratory distress syndrome.

Inflammatory bowel disease is a collective term for two similar diseasesreferred to as Crohn's disease (CD) and ulcerative colitis. CD is anidiopathic, chronic ulceroconstrictive inflammatory diseasecharacterized by sharply delimited and typically transmural involvementof all layers of the bowel wall by a granulomatous inflammatoryreaction. Any segment of the gastrointestinal tract, from the mouth tothe anus, may be involved, although the disease most commonly affectsthe terminal ileum and/or colon. Ulcerative colitis is an inflammatoryresponse limited largely to the colonic mucosa and submucosa.Lymphocytes and macrophages are numerous in lesions of inflammatorybowel disease and may contribute to inflammatory injury.

Asthma is a disease characterized by increased responsiveness of thetracheobronchial tree to various stimuli potentiating paroxysmalconstriction of the bronchial airways. The stimuli cause release ofvarious mediators of inflammation from IgE-coated mast cells includinghistamine, eosinophilic and neutrophilic chemotactic factors,leukotrines, prostaglandin and platelet activating factor. Release ofthese factors recruits basophils, eosinophils and neutrophils, whichcause inflammatory injury.

Atherosclerosis is a disease of arteries (e.g., coronary, carotid, aortaand iliac). The basic lesion, the atheroma, consists of a raised focalplaque within the intima, having a core of lipid and a covering fibrouscap. Atheromas compromise arterial blood flow and weaken affectedarteries. Myocardial and cerebral infarcts are a major consequence ofthis disease. Macrophages and leukocytes are recruited to atheromas andcontribute to inflammatory injury.

Rheumatoid arthritis is a chronic, relapsing inflammatory disease thatprimarily causes impairment and destruction of joints. Rheumatoidarthritis usually first affects the small joints of the hands and feetbut then may involve the wrists, elbows, ankles and knees. The arthritisresults from interaction of synovial cells with leukocytes thatinfiltrate from the circulation into the synovial lining of joints. Seee.g., Paul, Immunology (3d ed., Raven Press, 1993).

Another indication for chronic dosage of anti alpha-4 agents is intreatment of organ or graft rejection. Over recent years there has beena considerable improvement in the efficiency of surgical techniques fortransplanting tissues and organs such as skin, kidney, liver, heart,lung, pancreas and bone marrow. Perhaps the principal outstandingproblem is the lack of satisfactory agents for inducing immunotolerancein the recipient to the transplanted allograft or organ. When allogeneiccells or organs are transplanted into a host (i.e., the donor and doneeare different individuals from the same species), the host immune systemis likely to mount an immune response to foreign antigens in thetransplant (host-versus-graft disease) leading to destruction of thetransplanted tissue. CD8⁺ cells, CD4⁺ cells and monocytes are allinvolved in the rejection of transplant tissues. Antibodies directed toalpha-4 integrin are useful, inter alia, to block alloantigen-inducedimmune responses in the donee thereby preventing such cells fromparticipating in the destruction of the transplanted tissue or organ.See, e.g., Paul et al., 1996 Transplant International 9: 420-425;Georczynski et al., 1996 Immunol. 87: 573-580); Georcyznski et al., 1995Transplant. Immunol. 3: 55-61); Yang et al., 1995 Transplantation 60:71-76); Anderson et al., 1994 APMIS 102: 23-27.

A related use for anti alpha-4 agents is in modulating the immuneresponse involved in “graft versus host” disease (GVHD). See e.g.,Schlegel et al., J. Immunol. 155, 3856-3865 (1995). GVHD is apotentially fatal disease that occurs when immunologically competentcells are transferred to an allogeneic recipient. In this situation, thedonor's immunocompetent cells may attack tissues in the recipient.Tissues of the skin, gut epithelia and liver are frequent targets andmay be destroyed during the course of GVHD. The disease presents anespecially severe problem when immune tissue is being transplanted, suchas in bone marrow transplantation; but less severe GVHD has also beenreported in other cases as well, including heart and liver transplants.The therapeutic agents of the present invention are used, inter alia, toblock activation of the donor T-cells thereby interfering with theirability to lyse target cells in the host.

A further use of anti alpha-4 agents of the invention is inhibitingtumor metastasis. Several tumor cells have been reported to expressalpha-4 integrin and antibodies to alpha-4 integrin have been reportedto block adhesion of such cells to endothelial cells (Steinback et al.,1995 Urol. Res. 23: 175-83); Orosz et al., 1995 Int. J. Cancer 60:867-71); Freedman et al., 1994 Leuk. Lymphoma 13: 47-52); Okahara etal., 1994 Cancer Res. 54: 3233-6).

A further use of the anti-alpha-4 agents is in treating multiplesclerosis. Multiple sclerosis (MS) is a progressive neurologicalautoimmune disease that affects an estimated 250,000 to 350,000 peoplein the United States. Multiple sclerosis is thought to be the result ofa specific autoimmune reaction in which certain leukocytes attack andinitiate the destruction of myelin, the insulating sheath covering nervefibers. In an animal model for multiple sclerosis, murine monoclonalantibodies directed against alpha-4 beta-1 integrin have been shown toblock the adhesion of leukocytes to the endothelium, and thus preventinflammation of the central nervous system and subsequent paralysis inthe animals.

The onset of MS may be dramatic or so mild as to not cause a patient toseek medical attention. The most common symptoms include weakness (inone or more limbs, visual blurring due to optic neuritis, sensorydisturbances, diplopia and ataxia. The course of disease may bestratified into three general categories: (1) relapsing MS, (2) chronicprogressive MS, and (3) inactive MS. Relapsing MS is characterized byrecurrent attacks of neurologic dysfunction. MS attacks generally evolveover days to weeks and may be followed by complete, partial or norecovery. Recovery from attacks generally occurs within weeks to severalmonths from the peak of symptoms, although rarely some recovery maycontinue for 2 or more years.

Chronic progressive MS results in gradually progressive worseningwithout periods of stabilization or remission. This form develops inpatients with a prior history of relapsing MS, although in 20% ofpatients, no relapses can be recalled. Acute relapses also may occurduring the progressive course.

A third form is inactive MS. Inactive MS is characterized by fixedneurologic deficits of variable magnitude. Most patients with inactiveMS have an earlier history of relapsing MS.

The course of MS is also dependent on the age of the patient. Forexample, favourable prognostic factors include early onset (excludingchildhood), a relapsing course and little residual disability 5 yearsafter onset. By contrast, poor prognosis is associated with a late ageof onset (i.e., age 40 or older) and a progressive course. Thesevariables are interdependent, since chronic progressive MS tends tobegin at a later age that relapsing MS. Disability from chronicprogressive MS is usually due to progressive paraplegia or quadriplegiain individual patients. In one aspect of the invention, patients willpreferably be treated when the patient is in remission rather then in arelapsing stage of the disease.

Short-term use of either adrenocorticotropic hormone (ACTH) or oralcorticosteroids (e.g., oral prednisone or intravenousmethylprednisolone) is the only specific therapeutic measure fortreating patients with acute exacerbation of MS.

Newer therapies for MS include treating the patient with interferonbeta-1b, interferon beta-1a, and Copaxone® (formerly known as copolymer1). These three drugs have been shown to significantly reduce therelapse rate of the disease. These drugs are typically self-administeredintramuscularly or subcutaneously.

None of the currently available treatments inhibit demyelination or MS.One aspect of the invention contemplates treating MS with agentsdisclosed herein either alone or in combination with other standardtreatment modalities. Standard treatment modalities include but are notlimited to the following. Additional treatment modalities not discussedherein for use in treating MS in combination with the methods andcompositions disclosed herein depending on the state of disease in thepatient would be evident to the skilled practitioner. Such additionaltreatment modalities for MS other pathological inflammation wouldinclude other immunomodulators or immunosupressants.

The agents and pharmaceutical compositions discussed supra can bechronically administered for prophylactic and/or therapeutic treatmentsof the previously listed inflammatory disorders, including multiplesclerosis, inflammatory bowel disease, asthma, atherosclerosis,rheumatoid arthritis, organ or graft rejection and graft versus hostdisease. In therapeutic applications, compositions are administered to apatient suspected of, or already suffering from such a disease in anamount sufficient to cure, or at least partially arrest, the symptoms ofthe disease and its complications. An amount adequate to accomplish thisis defined as a therapeutically- or pharmaceutically-effective dose.

In prophylactic applications, pharmaceutical compositions arechronically administered to a patient susceptible to, or otherwise atrisk of, a particular disease in an amount sufficient to eliminate orreduce the risk or delay the outset of the disease. Such an amount isdefined to be a prophylactically effective dose. In patients withmultiple sclerosis in remission, risk may be assessed by NMR imaging or,in some cases, by presymptomatic indications observed by the patient.

Effective dosage regimes of the compositions of the present invention,for the treatment of the above described conditions will vary dependingupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicamentsadministered. Thus, treatment dosages will need to be titrated tooptimize safety and efficacy. In general, each administration of thedosage regime will range from about 0.0001 to 100 mg/kg, and moreusually 0.01 to 5 mg/kg of the host body weight. One preferred dosageregimen is 300 mg administered once per month for a period of at least 6months, more preferably 12 months and perhaps over the course of severalyears. Another dosage regimen that is preferred is a 3 mg per kilogramof patient weight per month. Such a regimen may be preferable forpediatric or adolescent patients in need of therapy.

Combination Therapies

The anti-alpha-4 agents of the invention can be used with effectiveamounts of other therapeutic agents against acute and chronicinflammation. Such agents include other antagonists of adhesionmolecules (e.g., other integrins, selectins, and immunoglobulin (Ig)super family members (see Springer, 1990 Nature 346: 425-433; Osborn,1990 Cell 62: 3; Hynes, 1992 Cell 9: 11). Integrins are heterodimerictransmembrane glycoproteins consisting of an α chain (120-180 kDa) and aβ chain (90-110 kDa), generally having short cytoplasmic domains. Forexample, three important integrins (i.e., LFA-1, Mac-1 and P150,95) havedifferent alpha subunits, designated CD11a, CD11b and CD11c, and acommon beta subunit designated CD18. LFA-1 (α_(L)β₂) is expressed onlymphocytes, granulocytes and monocytes, and binds predominantly to anIg-family member counter-receptor termed ICAM-1 and related ligands.ICAM-1 is expressed on many cells, including leukocytes and endothelialcells, and is up-regulated on vascular endothelium by cytokines such asTNF and IL-1. Mac-1 (α_(M)β₂) is distributed on neutrophils andmonocytes, and also binds to ICAM-1. The third β2 integrin, P150,95(α_(x)β₂), is also found on neutrophils and monocytes. The selectinsconsist of L-selectin, E-selectin and P-selectin.

Other anti-inflammatory agents that can be used in combination with theanti-alpha-4 agents include antibodies and other antagonists ofcytokines, such as interleukins IL-1 through IL-13, tumor necrosisfactors α and β, interferons α, β and γ, tumor growth factor beta(TGF-β), colony stimulating factor (CSF) and granulocyte monocyte colonystimulating factor (GM-CSF). Other anti-inflammatory agents includeantibodies and other antagonists of chemokines such as MCP-1, MIP-1α,MIP-1β, RANTES, exotaxin and IL-8. Other anti-inflammatory agentsinclude NSAIDS, steroids and other small molecule inhibitors ofinflammation. Formulations, routes of administration and effectiveconcentrations of agents for combined therapies are as described abovefor the humanized antibodies against alpha-4 integrin.

Additional agents for use in combination with agents which mediatealpha-4 integrin or dimers comprising alpha-4 integrin and treatinflammatory bowel disease (IBD), Crohn's Disease (CD) and ulcerativecolitis (UC), include but are not limited to 5-aminosalicylates,glucocorticoids, thioguanine derivatives, methotrexate (MTX),cyclosporine, antibiotics, and infliximab.

5-Aminosalicylates include sulfasalazine (also known as Azulfidine)which is a conjugate of mesalamine linked to sulfapyridine by a diazobond and is usually administered in an amount of 500 mg/day to about 6g/day. 5-Aminosalicylates can also be co-administered with aglucocorticoid. Preferably, a 5-aminosalicylate is used in combinationtherapy with one of the other agents discussed herein to treatulcerative colitis, however it can also be used to treat Crohn'sdisease. Non-sulfonamide containing formulations of mesalamine includebut are not limited to ASACOL®, CLAVERSA, SALOFALK, PENTASA®, DIPENTUM®,COLAZIDE and ROWASA®.

Glucocorticoids have been a mainstay of treatment for acute severeexacerbations of IBD since 1955, when they first where shown to beefficacious in UC. Oral prednisone can be administered in conjunctionwith any of the agents disclosed herein. Typically, 20 to 40 mg of oralprednisone is administered once a day. Glucocorticoids can also beadministered intravenously and via enemas in combination with orconcurrently with or within a short time before/after an anti-alpha-4integrin agent is administered. For example, hydrocortisone is availableas a retention enema (100 mg/GO mL) and the usual dose is one 60-mLenema per night for 2 to 3 weeks. This can be altered when used incombination with the therapies and agents discussed herein as would beunderstood by the artisan of ordinary skill. Other steroids that can beused include, but are not limited to, prednisolone methasulfobenzoate,tixocortol pivalate, fluticasone propionate, beclomethasonedipropionate, and budesonide.

Thioguanine derivatives are also useful in the treatment of IBD, CD andUC. These include but are not limited to 6-mercaptopurine (6-MP) andazathioprine (IMURAN). The two drugs can be used interchangeably incombination with any of the alpha-4 integrin modulating agents discussedherein.

Methotrexate (MTX) is also contemplated for use in combination with thealpha-4 integrin regulatory agents discussed herein. Preferably, MTX isadministered via intramuscular injection (i.m.) to the subject incombination with an anti-alpha-4 integrin agent. MTX is effective insteroid-dependent CD, but not as useful in UC. MTX can be administeredin amounts of about 15 to about 25 mg per week per subject or asnecessary as determined by the artisan of ordinary skill.

Cyclosporines (e.g., SANDIMMUNE®, NEORAL®) can also be used incombination with the alpha-4 integrin modulating agents discussed hereinto treat pathological inflammation of the bowel. This can be used totreat acute, severe UC, which does not respond to glucocorticoids.

Infliximab (i.e., REMICADE®) can also be used to treat CD in combinationwith the alpha-4 integrin modulating agents indicated herein. Infliximabis an immunoglobulin that binds to TNF and thereby neutralizes itsactivity. Other anti-TNF antibodies, such as CDP571, can also be used incombination with the alpha-4 integrin modulating agents disclosedherein.

Antibiotics are also contemplated for use in combination with thealpha-4 integrin modulating agents indicated herein to modulate UC, IBDand CD. For example, patients can be treated with metronidazole orciprofloxacin (or pharmacological equivalents thereof) in combinationwith an alpha-4 integrin mediating agent or in the form of an admixture.

Also contemplated is the use of supportive therapies for IBD, CD and UCin conjunction with agents that mediate alpha-4 integrin or dimerscomprising alpha-4 integrin and treat inflammatory bowel disease,Crohn's Disease and ulcerative colitis. Supportive therapies include,but are not limited to, analgesics, anticholinergic and antidiarrhealagents. Combining such supportive therapies can be useful in thebeginning of a treatment regimen in reducing a patient's symptoms andimproving their quality of life. Supportive therapies includeadministering oral iron, folate, and vitamin B₁₂. Antidiarrheal agentsinclude, but are not limited to diphenoxylate, codeine, loperamide, andanticholinergics (or pharmacological equivalents thereof), which can beadministered to patients with mild disease to reduce the frequency ofbowel movements and relive rectal urgency. Cholestyramine can be used inpatients to prevent bile salt-induced colonic secretion in patients whohave already undergone limited ileocolic resections prior to treatmentwith the chronic regimens described herein. Anticholinergic agentsinclude, but are not limited to, clidinium bromide, dicyclominehydrochloride, tincture of belladonna and the like, and are useful toreduce abdominal cramps, pain and rectal urgency.

For treatment of MS, the anti-alpha-4 integrin agents (e.g.,anti-alpha-4 integrin antibodies, small compound alpha-4 integrinantagonists and the like) can be combined with other compounds orcompositions used to treat, ameliorate or palliate symptoms associatedwith MS.

Other agents utilized to treat, ameliorate or palliate symptomsassociated with MS, include but are not limited to: muscle relaxants(e.g., Diazepam, cyclobenzaprine, Clonazepam, clonidine, primidone, andthe like), anticholinergics (e.g., propantheline, dicyclomine, and thelike), central nervous system stimulants (e.g., Pemoline), non-steroidalanti-inflammatory agents (NS such as ibuprofen, naproxen andketoprofen), interferons, immune globulin, glatiramer (Copaxone®),mitoxantrone (Novantrone®), misoprostol, tumor necrosis factor-alphainhibitors (e.g., Pirfenidone, infliximab and the like) andcorticosteroids (e.g., glucocorticoids and mineralocorticoids).

Common agents for treating multiple sclerosis include interferon beta-1b(Betaseron®), interferon beta-1a (Avonex®), high-dose interferon beta-1a(Rebif), Glatiramer (Copaxone®), immune globulin, mitoxantrone(Novantrone®), corticosteroids (e.g., prednisone, methylprednisolone,dexamethasone and the like). Other corticosteroids may also be used andinclude but are not limited to cortisol, cortisone, fludrocortisone,prednisolone, 6α-methylprednisolone, triamcinolone, and betamethasone.

Dosage forms of the agents to be used in combination with the compoundsand compositions disclosed herein would vary depending on the subjectand drug combination being utilized. For example, interferons aretypically administered as follows: Interferon beta-1a (Avonex®) isadministered 30 μg once a week; interferon beta-1a is administered atabout 22 μg or 44 μg three times a week; and interferon beta-1b(Betaseron®) is administered at 250 μg on alternate days (Durelli etal., Lancet 359: 1453-60, 2002). Typically the interferons areadministered for relapsing or remitting multiple sclerosis. Thus incombination with the anti-alpha-4 integrin agents disclosed herein,preferred ranges of interferons may include about 0.1 μg to about 250 μgand more preferably about 0.5 μg to about 50 μg, depending on the mannerin which the agent is administered in conjunction with the otheranti-alpha-4 integrin compounds and compositions disclosed herein.

NS or NSAIDs contemplated for use with this invention include but arenot limited to non-selective COX inhibitors and selective COX-2inhibitors. Non-selective COX inhibitors include but are not limited tosalicylic acid derivatives (e.g., aspirin, sodium salicylates, cholinemagnesium trisalicylate, salsalate, diflunisal, sulfasalazine, andolsalazine), para-aminophenol derivatives (e.g., acetaminophen), indoleand indene acetic acids (e.g., tolmetin, diclofenac, and ketorolac),heteroaryl acetic acids (e.g., abuprofen, naproxen, flurbiprofen,ketoprofen, fenprofen, and oxaprozin), anthranilic acids or fenamates(e.g., mefenamic acid and meclofenamic acid), enolic acids (e.g.,oxicams such as piroxicam and meloxicam), and alkanones (e.g.,nabumetone). Selective COX-2 inhibitors include diaryl-substitutedfuranoses (e.g., rofecoxib), diaryl-substituted pyrazoles (e.g.,celecoxib), indole acetic acids (e.g., etodolac), and sulfonanilides(e.g., nimesulide). NS are oftentimes administered in combination withinterferon to lessen the flu-like symptoms experienced by patientsreceiving, for example, Avonex®. Common NS agents include naproxen,ibuprofen and ketoprofen. Paracetamol is also frequently administered topatients. See, Reess et al., 2002 Mult. Scler. 8: 15-8.

Glatiramer acetate (GA, Copaxone®) is a synthetic molecule that inhibitsactivation of myelin basic protein-reactive T cells and induces a T-cellrepertoire characterized by anti-inflammatory effects. Moreover,Glatiramer can access the central nervous system (CNS), whereasinterferon-beta cannot (Dhib-Jalbut, 2002 Neurology 58: S3-9;Weinstock-Guttman et al., 2000 Drugs 59: 401-10).

Mitoxantrone is an anthracenedione synthetic agent, which has been shownto be effective for treating secondary progressive multiple sclerosis(SP-MS). However, use of this drug is again limited by its cumulativecardiotoxicity (Weinstock-Guttman et al., 2000).

Tumor necrosis factor-alpha (TNF-α) may be a key cytokine indemyelination (Walker et al., 2001 Mult. Scler. 7: 305-12). Thus use ofagents that antagonize TNF-α function or inhibit its synthesis may beuseful in combination with the agents and compounds disclosed herein.This can include anti-TNF-α antibodies (e.g., infliximab) as well asagents such as pirfenidone. Pirfenidone is a non-peptide drug, which hasbeen shown to decrease synthesis of TNF-α and to block receptors forTNF-α. Id.

The long mainstay in most demyelinating conditions and diseases has beenthe use of ACTH, glucocorticoids and corticoid steroids. These agentsare used for their anti-edema and anti-inflammatory effects. ACTH iscommonly administered to a subject at 80 U given intravenously in 500 mLof 5% dextrose and water over 6-8 hours for 3 days. It may also beadministered at 40 U/ml intramuscularly at a dose of 40 U every 12 hoursfor 7 days, with the dose then reduced every 3 days. See, S. Hauser,“Multiple sclerosis and other demyelinating diseases,” in Harrison'sPrinciples of Internal Medicine 2287-95 (13^(th) ed., Isselbacher etal., ed. 1994). Methylprednisolone is typically administered slowly in500 ml D5W over 6 hours, preferably in the morning. Common dosagesinclude 1000 mg daily for 3 days, 500 mg daily for 3 days and 250 mgdaily for 3 days. Id. A methylprednisolone-prednisone combination isalso commonly administered. Typically about 1,000 mg of intravenousmethylprednisolone is administered over three days followed by oralprednisone at 1 mg/kg per day for 14 days. Thus, for use in combinationwith the compounds and compositions disclosed herein, the steroids maybe administered in amounts ranging from about 1 to about 1,000 mg/kgover about 1 to 14 days, as needed.

A side effect in demyelinating conditions such as MS, is fatigue anddecreased cognitive function. Agents such as amantadine hydrochlorideand pemoline have been frequently used to treat fatigue associated withMS (Geisler et al., 1996 Arch. Neurol. 53: 185-8).

The benefit of such combination therapies is that it may lessen theclass-specific and agent-specific side effects currently encounteredwith some of the drugs. Class-specific side effects of interferon-betainclude fever, chills, myalgias, arthralgias and other flu-like symptomsbeginning 2-6 hours after injection and typically resolving 24 hourspost injection. Occasionally interferon-beta also induces transientworsening of preexisting MS symptoms. Agent specific side effectsinclude injection site reactions with interferon beta-1b. Management ofthese effects can be accomplished by tailoring the dose and time ofadministration, prescribing appropriate combinations of acetaminophen,non-steroidal anti-inflammatory drugs (NS or NSAIDS) and steroids. SeeMunschauer et al., 1997 Clin. Ther. 19: 883-93.

Thus, combinations of drugs that can lessen the quantity of a particulardrug administered may reduce adverse side effects experienced by apatient.

When administered in combination, the small compound alpha-4-integrinantagonists may be administered in the same formulation as these othercompounds or compositions, or in a separate formulation. Whenadministered in combination, the anti-alpha-4-antibodies are generallyadministered in a separate formulation than the other compounds andcompositions. When administered in combinations, the anti-alpha-4 agentsmay be administered prior to, following, or concurrently with the othercompounds and compositions used to treat, ameliorate, or palliatesymptoms.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description ofrepresentative examples of how to make and use embodiments of thepresent invention, and are not intended to limit the scope of what theinventors regard as their invention, nor are they intended to representthat the experiments below are all or the only experiments performed.Efforts have been made to ensure accuracy with respect to numbers used(e.g., amounts, temperature, etc.) but some experimental errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, molecular weight is weight average molecularweight, temperature is in degrees Centigrade, and pressure is at or nearatmospheric.

Example 1 Controlled Trial of Natalizumab in Relapsing MultipleSclerosis

Patient Population

Twenty-six clinical centers in the United States, Canada, and the UnitedKingdom enrolled 213 patients from September 1999 until May 2000. Theinstitutional review board or central and local ethics committeeapproved the protocol. All patients gave written informed consent. Studyoversight was provided by an independent safety data monitoringcommittee.

Eligible subjects were required to be age 18 through 65 years, withPoser criteria defined clinically or laboratory supported definite MS,either relapsing-remitting or secondary progressive, (Poser et al., 1983Ann. Neurol. 13: 227-31; Lublin et al., 1996 Neurology 46: 907-11), ahistory of at least two relapses within the previous two years, abase-line Kurtzke Expanded Disability Status Score (EDSS) (Kurtzke, 1983Neurology 33: 1444-52) between 2 and 6.5, and a minimum of three lesionson T₂-weighted brain MRI. Patients were excluded if they receivedimmunosuppressive or immunomodulating treatments within the past 3months, or experienced a relapse, or received systemic corticosteroidswithin the past 30 days.

Study Design and Randomization

Patients were randomly assigned to one of three treatment groups: 3mg/kg natalizumab, 6 mg/kg natalizumab, or placebo according to acomputer-generated block randomization schedule. Patients received sixintravenous infusions at 28-day intervals and then had six months ofsafety follow-up. The investigator, all other study personnel, andpatients were blinded to treatment assignment.

Study Procedures and Endpoints

Unenhanced proton density T₂-weighted and Gd-enhanced T₁-weighted MRIbrain scans were obtained during the screening phase (month-1),immediately before each treatment (month 0-5), and one month after thelast treatment (month 6). Follow-up MRI scans were obtained at months 9and 12. Forty-six contiguous, 3-mm thick, axial slices through the brainwere acquired. MRI analysis was performed by a single center blinded topatient treatment and history. Lesions were identified on hard copyimages by two experienced clinicians working by consensus.

The prospective primary outcome measure was the number of newGd-enhancing lesions over the 6-month treatment period, defined as theperiod following the first infusion to one month after the lastinfusion. Other MRI parameters evaluated included: the number ofpersistent Gd-enhancing lesions (enhancing lesions that had alsoenhanced on the previous monthly scan); the volume of Gd-enhancinglesions (measured by a semiautomated local thresholding method; Grimaudet al., 1996 Magn. Reson. Imaging 14: 495-505); the number of new activelesions (i.e., new Gd-enhancing lesions plus new or enlarging,non-enhancing T2 lesions); and the number of active scans (i.e.,containing one or more new Gd-enhancing lesions).

Clinical endpoints included relapse frequency and changes in EDSS, and aself-reported global assessment using a visual-analog scale (VAS). Alladverse events were recorded. Patients were examined at scheduledquarterly intervals, and at unscheduled visits for suspected relapses,by the treating and evaluating neurologists who were both unaware of thepatient's treatment assignment. The treating neurologist performed amedical history and examination, and recorded adverse events. Theevaluating neurologist assessed neurological status and assigned an EDSSscore without knowledge of the patient's history or prior EDSS scores.

An objective relapse was defined as the occurrence of an acute episodeof new or worsening MS symptoms lasting at least 48 hours following astable period of at least 30 days. It was also accompanied by anincrease of at least one point in the EDSS score, an increase of atleast one point on two functional system scores (FSS), or an increase ofat least two points on one FSS compared with base-line, as determined bythe evaluating neurologist. Neurological symptoms that did not meet theabove criteria for relapse, but were assessed by the treatingneurologist to constitute a relapse, were also recorded (totalrelapses).

On a visual analog scale (VAS), patients marked a location along a 10-cmline that reflected their assessment of their overall well-being atbase-line and after 3 and 6 months treatment, with higher scoresreflecting greater well-being.

Patients were followed clinically to month 12. Patients who discontinuedtreatment, but who did not reach the endpoint, were encouraged to returnfor follow-up assessments.

Statistical Analysis

Sample size estimates were based on the number of new Gd-enhancinglesions observed during the first 12 weeks following the first infusionin a previous clinical trial of natalizumab (Tubridy et al., 1999Neurology 53: 466-72). Based on the results of this previous trial, andusing sample size methodology appropriate for a two-sided, two-groupcomparison at the 5 percent level of significance, based on theWilcoxon-Mann-Whitney statistic (Noether, 1987 J. Amer. Stat. Assoc. 82:645-7), it was calculated that approximately 73 patients were needed ineach group for 80 percent power.

The primary comparison of the number of Gd-enhancing lesions between 6mg/kg natalizumab and placebo as well as Gd-enhancing volumes wereevaluated with the Wilcoxon-Mann-Whitney rank-sum test. Missing valuesdue to one or more MRI scans not being performed were imputed byreplacing the missing value with the average number of lesions onavailable scans for that patient. MRI scans obtained from patients whoreceived systemic corticosteroids within the previous 30 days werediscarded and treated as missing values. The Cochran-Mantel-Haenszelcorrelation statistic, using equally spaced scores for the groups andrank scores for the primary outcome variable, was used to test for adose-response relationship using data from all three groups.

Pearson's chi-square test was used to compare proportions of patientswith relapses. Changes from baseline in EDSS and VAS were analyzed usinga two-way ANOVA with study center and treatment groups as independentvariables.

All analyses included all randomized patients and followed theintention-to-treat principle. All reported P values are two-tailed.There were no significant differences in demographic characteristics, MSdisease history, entry EDSS, and MRI parameters among the three groupsat base-line (Table 1).

TABLE 1 DEMOGRAPHIC AND BASELINE CHARACTERISTICS OF RANDOMIZED PATIENTSNatalizutuab PLACEBO 3 MG/KG 6 MG/KG CHARACTERISTIC (N = 71) (N = 68) (N= 74) Age, years Mean 42.9 42.8 44.9 Range 22-66 22-65 30-63 Gender N(%) Male 25 (35.2) 21 (30.9) 15 (20.3) Female 46 (64.8) 47 (69.1) 59(79.7) MS category N (%) R-R 45 (63.4) 47 (69.1) 52 (70.3) S-P 26 (36.6)21 (30.9) 22 (29.7) EDSS Mean 4.40 4.21 4.32 Range 2.0-6.5 1.0-6.50.0-6.5 Disease duration Mean 10.2 11.6 13.1 Years Range  1-32  0-40 2-39 Number of relapses Mean 3 2.9 3.1 In past 2 years Range  2-12 2-10 2-8 Time since last relapse Mean 6.5 7.2 6 Months Range  2-17 2-24  2-22 Screening T₁-weighted MRI (Month 1) Number (%) of scans withone or 28 (40)   29 (43)   29 (40)   more Gd-enhancing lesion(s) Numberof Gd-enhancing brain lesions Mean 1.6 1.5 1.7 Range  0-42  0-18  0-23Baseline T₁-weighted MRI (Month 0) Number (%) of scans with one or 22(31)   29 (43)   32 (43)   more new Gd-enhancing lesion(s) Number of newGd-enhancing brain lesions Mean 1.3 1.3 1.4 Range  0-28  0-32  0-12Primary Outcome

Patients in the placebo group exhibited an average of 9.6 newGd-enhancing lesions during the six-month treatment period. Thecorresponding values in the groups receiving natalizumab were 0.7 forthe 3 mg/kg group (P<0.0001) and 1.1 for the 6 mg/kg group(P<0.0001)(see Table 2). This difference constituted a 93% and an 88%reduction in new Gd-enhancing lesions in the 3 mg/kg and 6 mg/kg groups,respectively. A difference between treatment groups compared withplacebo was apparent after the first infusion (FIG. 1).

TABLE 2 SUMMARY OF MRI ACTIVITY DURING TREATMENT (MONTHS 1-6) ANDFOLLOW-UP (MONTHS 9 AND 12) 3 MG/ 6 MG/ P PLACEBO KG KG VALUE* Newenhancing lesions M1- Mean 9.6 0.7 1.1 (i) <0.0001 Median 2.0 0 0 (ii)<0.0001 SD 27.4 2.1 2.7 Persistent enhancing lesions M1-6 Mean 3.6 0.81.3 <0.0001 Median 1 0 0 SD 6.5 1.9 2.6 New active lesions M1-6 Mean 9.70.8 1.1 <0.0001 Median 2.0 0 0 SD 27.4 2.2 3 Active scans M1-6 (%) 39% 9% 11% (i) <0.0001 (ii) <0.0001 Enhancing lesion volume M1-6(mm³) Mean1169.0 156 279.0 (i) 0.005 Median 266 0 0 (ii) 0.01 SD 2666 359.0 632.0New enhancing lesions M9 and 12. Mean 2.5 2.6 2.1 (i) 0.90 Median 1.00.5 0 (ii) 0.59 SD 4.37 4.58 4.96 Persistent enhancing lesions M9 and 12Mean 0.2 0.1 0.1 0.029 Median 0 0 0 SD 0.64 0.32 0.3 New active lesionsM9 and 12 Mean 2.7 2.8 2.3 0.424 Median 1.0 0.5 1.0 SD 4.49 5.69 5.11Active scans M9 and 12 42% 40% 35% (%) Enhancing lesion volume M9 and12(mm³) Mean 427.0 323 233 0.260 Median 88.0 31.0 0 SD 797.0 591.0 686*(i) comparison of placebo vs. 3 mg/kg natalizumab; (ii) comparisonplacebo vs. 6 mg/kg natalizumab.Secondary MRI Outcomes

There was a significant and marked reduction in the cumulative number ofpersistent enhancing lesions, new active lesions, total volume ofenhancing lesions, and percentage of active scans from months 1-6 (Table2; FIG. 2).

Clinical Efficacy Outcomes

During the six month treatment period, a total of 35 relapses werereported in 26 of the 71 placebo patients; 18 relapses were reported in13 of the 68 patients receiving 3 mg/kg natalizumab, and 15 relapseswere reported in 14 of the 74 patients receiving 6 mg/kg (P=0.05,placebo vs. all natalizumab-treated patients). Applying the morestringent objective relapse criteria, the effect was equally strong: 18relapses in 15 placebo patients; 3 relapses in 3 patients receiving 3mg/kg natalizumab; 8 relapses in 8 patients receiving 6 mg/kgnatalizumab (P=0.05). More relapses in the placebo group requiredsteroid treatment than in the treated arms (22 in placebo, 5 in the 3mg/kg natalizumab, and 7 in the 6 mg/kg natalizumab groups, P=0.007,placebo vs. all natalizumab-treated patients).

On the VAS, patients in the placebo group reported no change, whilethose in the natalizumab groups reported an improvement in well-being bymonth 6, at which time the difference between groups was significant(P=0.033, placebo vs. all natalizumab-treated patients). No significantchanges in EDSS were observed in any group during treatment.

Antibody Concentration and Receptor Saturation

Serum samples were collected from patients at each visit and analyzedquantitatively for antibodies directed specifically against natalizumabusing an enzyme linked immunosorbant assay (ELISA). Natalizumab serumlevels and receptor occupancy by natalizumab were also measured in asubgroup of 12 to 14 patients per treatment group before each infusionand at 2 hours, 24 hours, 1, 2, and 3 weeks after the first and lastinfusions.

Serum antibody concentrations were determined using an ELISA assay. Inbrief, a 2.0 μg/mL solution of a capture antibody that bindsspecifically to natalizumab was prepared in a solution containing sodiumbicarbonate to pH 8.3. 100 μL of the antibody solution was added to eachwell of a Costar 96-well microtiter plate. The plate was covered withplate sealing tape and incubated at ambient temperature for 12-26 hours.The plate was aspirated, 200 μL of blocking buffer (0.25% casein in PBS,pH 7.4) added to each well, and incubated for an additional hour atambient temperature. Plates were then either dessicated and stored forlater use, or washed once with 300 μL wash buffer (TBS, pH 7.5containing 0.05% Tween-20). If plates were dessicated, they wererehydrated just prior to use by adding 300 μL wash buffer to each welland incubating for 1-2 minutes. Plates were aspirated and inverted ontotissue paper to absorb excess moisture.

Test samples were diluted in casein diluent prior to the ELISA (0.25%casein in PBS, with 0.05% Tween 20, pH 7.4). Typically, two or threedilutions of each sample are tested to ensure the natalizumab valueswere accurate. Dilution control samples were also prepared using knownquantities of natalizumab to monitor the accuracy of the remainingsteps.

100 μL of reference standard, test sample, or dilution control samplewas added to each well, and the plate incubated between 60-75 minutes atambient temperature. The plates were washed three times with wash bufferand remaining moisture removed. 100 μL freshly diluted mouse anti-humanIgG4-alkaline phosphatase conjugate was added to each well, and theplates were incubated for an additional 60 minutes at ambienttemperature. Following incubation, plates were washed four times withwash buffer, and remaining moisture removed. 100 μL fluorescentsubstrate A was added using a calibrated multichannel pipetter, and theplates incubated 45-60 minutes at ambient temperature. The levels ineach well were determined using an fmax Fluorescence Microplate Readerusing the SOFTmax Pro Version 1.3.1 protocol file conc102.ppr.

The results are as shown in FIGS. 3 and 4. As shown in these figures,levels of natalizumab decreased between dosings, and by month 7 or 8(i.e., 2 to 3 months following the final dosing) the antibody wasundetectable in the majority of patients.

As well as measuring serum antibody concentration, levels of receptorsaturation, and specifically levels of VLA-4 saturation, were determinedduring the dosing study using FACS analysis. Determination of VLA-4saturation was determined by an indirect immunoassay using flowcytometry.

Approximately 1 mL of a blood sample from a patient was aliquoted intotwo 15 mL polypropylene tubes, and cold wash buffer (human serum(Scantibodies Laboratory, Inc. Part 3SH341) diluted to 3% in PBS) addedto the 14 mL mark on the tube. The tubes are centrifuged at 2,200 rpmfor 5 minutes at 10-15° C., and the liquid aspirated and discarded. Thecell pellet is resuspended in cold wash buffer to a total of 1 mL.

500 μg/mL of a natalizumab stock reference standard is prepared in washbuffer. 20 μL of the diluted natalizumab stock reference was added tothe first tube, and 20 μL wash buffer was added to the second tube. Bothtubes were incubated on ice for 30 minutes in the dark. Afterincubation, the tubes were filled with wash buffer up to the 14 mL mark,and centrifuged at 2,200 rpm for 5 minutes in the cold. The supernatantwas aspirated, and the wash step repeated a second time. Following thesecond wash, the cells were resuspended in wash buffer to the 1 mL mark.

10 μL of R-Phycoerythrin (PE)-conjugated anti-human CDw49d antibody(Pharmingen, Cat. 431475X) was added to each tube and the tubes vortexedgently. The tubes were incubated at 2-8° C. for 10-15 minutes in thedark. Following incubation, 2 mL 1× lysing solution, pH 7.4, was addedand each tube gently vortexed. The tubes were incubated on ice for 10-15minutes at 2-8° C., centrifuged 5 minutes in the cold, and thesupernatant aspirated from the cell pellet. The cell pellet wasresuspended in 4 mL cold staining buffer, and the tubes centrifugedagain at 2,200 rpm in the cold. The supernatant was then very carefullyremoved, 0.5 mL of a fixative solution (Ortho's Fixative pH 7.6) added,and the tubes immediately vortexed to ensure that cells were resuspendedin the fixative. The tubes were covered with aluminum foil until FACSassay.

Each sample was then for VLA-4 receptor saturation in the samples withand without analyzed natalizumab using the FACS Calibur flow cytometerand CellQuest™ software. The CellQuest™ software allows acquisition andanalysis of data from the flow cytometer.

Receptor saturation levels are shown in FIG. 5. The levels of receptorsaturation for months 1-4 were determined prior to that month's dosing.The levels of receptor saturation produced by one month dosage intervalswere consistently fairly high, and these chronic levels were sufficientto maintain a suppression of the pathological inflammation andassociated physiological hallmarks of the disease. On average, thelevels of saturation were maintained for a month at a mean of at least67% and a median of at least 75%. These levels were sufficient for thesuppression of brain lesions in the treated patients (see FIG. 1). Theminimum level of saturation determined in the study preinfusion frommonth 2 to month 5 (and week 21 following administration at month 5) isparticularly low as compared to the mean and median values due to asingle patient with an antibody response to natalizumab.

As the receptor saturation levels dropped in the patients, so did theefficacy of the treatment. Mean receptor saturation levels of 42% andmedian receptor saturation levels of 41% receptor saturation in thetreated population were not associated with suppression of brain lesionsin the patient population (See FIGS. 2 and 5), and thus a chronicreceptor saturation level above this is necessary for effectivesuppression of pathological inflammation using agents such as alpha-4inhibitors.

Safety and Tolerability

Repeated natalizumab treatment, at either the 3 or 6 mg/kg dose,appeared well tolerated by patients with MS over a six month treatmentperiod. Similar numbers of patients from each group experiencedtreatment-emergent adverse events. Though not significant, certainadverse events occurred more commonly with natalizumab compared toplacebo (Table 3). A sustained mild lymphocytosis was seen in thenatalizumab arms over the six month treatment period.

TABLE 3 ADVERSE EVENTS REPORTED MORE COMMONLY IN NATALIZUMAB- TREATEDPATIENTS VERSUS PLACEBO* PLACEBO 3 MG/ 6 MG/ (71) KG (68) KG (74) Totalnumber of patients 68 (96%) 62 (91%) 70 (95%) with adverse events Bodyas whole Infection 10 (14%) 14 (21%) 14 (19%) Digestive systemFlatulence  0  4 (6%)  0 (0%) Nervous system Circumoral parasthesia  1(1.0%)  5 (7%)  2 (3%) Respiratory system Sinusitis  3 (4%)  7 (10%)  3(4%) Pharyngitis  8 (11%) 10 (15%) 15 (20%) Skin and appendages Rash  4(6%)  6 (9%)  8 (11%) Urogenital system Infection 10 (14%) 14 (21%) 11(15%) *To be included in the Table, a difference of at least 5 percentin the incidence of adverse events was required between the placebo armand one of the natalizumab arms.

There were no significant differences in the number of serious adverseevents (SAEs) reported in the placebo and treatment arms (i.e., 7placebo-treated patients reported 11 SAEs, 5 patients receivingnatalizumab 3 mg/kg reported 5 SAEs, and 3 patients receivingnatalizumab 6 mg/kg reported 4 SAEs). Of these, four were considered tobe immune-mediated and related to study drug. There was one anaphylacticreaction with urticaria and bronchospasm in the 3 mg/kg group, which wasrapidly reversed with antihistamines and steroid treatment. There werethree reports of serum sickness, one in each group including placebo.Only one event was accompanied by a change in complement levels and alloccurred at the same investigative site. Overall, these eventscomplicated fewer than one in 250 infusions.

There were no differences in the number of patients that discontinuedtreatment due to an adverse event between groups (i.e., 3 in the placebogroup, 4 in the 3 mg/kg group and 3 in the 6 mg/kg group). There was onedeath in the study secondary to pleural carcinomatosis complicated byhemothorax in a placebo patient.

Rate of antibody formation was also assessed. Overall, 15natalizumab-treated patients (11 percent) developed anti-natalizumabantibodies: 13 during the treatment period, and 2 during thepost-treatment follow-up period. The clinical relevance, if any, of thepresence of anti-natalizumab antibodies is not currently known.

Maximal serum concentrations of natalizumab were dose dependent andthere was no significant accumulation observed with repeated dosing. Thepatients receiving natalizumab at 3 mg/kg exhibited greater than 80%saturation of the VLA-4 receptor during the treatment period; receptoroccupancy was higher (approximately 90%) and more prolonged in patientsreceiving natalizumab 6 mg/kg.

Post-Treatment Follow-Up: Months 6-12

The cumulative number of new enhancing lesions and active scans (months9 and 12 combined) were similar in all three groups (Table 2). There wasa trend to less activity in the 6 mg/kg group at month 9. There was nosignificant difference in the total number of reported clinical relapsesbetween the three groups: 24 in the placebo group, 24 in the 3 mg/kggroup, and 26 in the 6 mg/kg group or in the number of relapses asdetermined by the predefined objective criteria.

This study is the first to provide strong MRI and clinical evidence inhumans that selective inhibition of alpha-4 integrin-mediated leukocyteadhesion and trafficking is an effective approach to the chronictreatment of MS. Both dose levels of the alpha-4 integrin-specifichumanized monoclonal antibody natalizumab demonstrated highlystatistically significant effects compared with placebo on suppressionof new Gd-enhancing inflammatory brain lesions in patients with MS overthe six month treatment period. A reduction of these lesions innatalizumab-treated patients was observed one month after the firstinfusion and was sustained throughout the treatment period. For bothdose levels, the reduction was approximately 90 percent, an effectgreater than the 50 to 70 percent reduction reported withbeta-interferons (MS/MRI Analysis Group, 1995 Neurology 4: 1277-1285;Jacobs et al., 1996 Ann. Neurol. 39: 285-294; and PRISMS (Prevention ofRelapses and Disability by Interferon beta-1a Subcutaneously in MultipleSclerosis) Study Group, 1998 Lancet 352: 1498-1504).

Moreover, the effects of natalizumab on MRI outcomes in this trial aresupported by clinical observations. This study was not prospectivelypowered to show effects on clinical outcomes. Nonetheless, treatmentwith natalizumab resulted in a significant reduction in relapse rate anda perception of increased well-being among patients. When all reportedclinical relapses are considered, both natalizumab groups experiencedsignificantly fewer relapses than the placebo group during six months oftreatment. A significant reduction in these episodes was also observedusing predefined relapse criteria; a more stringent measure, because itrequires a change in objective signs. The effect of natalizumab onrelapses exceeds that of the currently approved treatments for MS, whichdisplay approximately 30 percent effect (MS/MRI Analysis Group, supra;Jacobs et al., supra; PRISMS Study Group, supra; Johnson et al., 1995Neurology 45: 1268-76).

Importantly, no rebound effects on new MRI lesions or relapses wereobserved in the natalizumab groups after termination of treatment.Further, monthly infusions of natalizumab for six months were welltolerated, and associated with a safety profile similar to placebo andacceptable for chronic treatment of MS.

The results of this study provide further support for the role ofalpha-4 integrin, and the immune cells that express it, in thepathogenesis of acute inflammatory brain lesions in patients with MS.The reduction in new Gd-enhancing lesions was evident after one month oftreatment. This observation suggests natalizumab acts early in lesiondevelopment by preventing the appearance of new lesions.

In summary, natalizumab has demonstrated strong effects on clinicallymeaningful parameters in this placebo-controlled trial in patients withrelapsing MS. Therapy was well tolerated during this six-month trial.The beneficial effects of natalizumab on the appearance of newinflammatory CNS lesions, the occurrence of clinical relapses andimprovement in patient well-being observed in this study indicate thepotential for observing effects on disability in the longer term studiescurrently in progress.

Example 2 Controlled Trial of Natalizumabin Crohn's Disease

Methods

In a double-blind, placebo-controlled trial, 248 patients with moderateto severely active Crohn's disease (CD) were randomized to receive twoinfusions of placebo or infusion of natalizumab at 3 mg/kg followed byplacebo; or two infusions of natalizumab at 3 mg/kg or 6 mg/kg, at a4-week interval. Outcome measures included the Crohn's Disease ActivityIndex (CDAI), health-related quality of life (QOL), and serum C-reactiveprotein levels.

Natalizumab increased the rates of clinical remission and clinicalresponse, and improved QOL in patients with active CD, whiledemonstrating a safety profile acceptable for treatment of this disease.

Patient Population

After receiving approval from the local ethics committee, each centerscreened male and female patients of at least 18 years of age who hadclinical evidence of moderate to severely active CD defined as a CDAI ofat least 220 but less than or equal to 450. Of 311 patients screened,248 were randomized at thirty-five study centers in Belgium, the CzechRepublic, Denmark, Germany, Israel, the Netherlands, Sweden, and theUnited Kingdom from September 1999 to August 2000. All patients gaveinformed, written consent. Patients who received methotrexate,cyclosporin, or any investigational agents within 3 months wereexcluded; patients receiving azathioprine or 6-mercaptopurine wererequired to have been on a stable dose for at least 4 months. Otherexclusions included prior antibody treatment; current use of oralprednisolone at a dose greater than 25 mg/day; current use of anelemental diet or parenteral nutrition; infectious or neoplasticdiseases of the bowel; bowel surgery within 3 months; presence of acolostomy, ileostomy, or a colorectostomy with ileorectal anastomosis;symptoms due mainly to the presence of fibrotic strictures; and clinicalimpression that the patient was likely in the near term to requireemergency abdominal surgery.

Study Design and Randomization

Eligible patients were randomly assigned to one of four treatmentregimens according to a computer-generated block randomization schedule.Each group received two intravenous infusions spaced by a 4-weekinterval. The four treatment regimens were two infusions of placebo; oneinfusion of natalizumab at 3 mg/kg followed by placebo infusion; and twoinfusions of natalizumab at 3 or 6 mg/kg. The investigator, all otherstudy personnel, and patients were blinded to treatment assignment.

Study Procedures and Endpoints

The primary efficacy endpoint was the proportion of patients inremission (CDAI<150) at week 6. The CDAI incorporates eight relatedvariables: number of liquid or very soft stools per day, severity ofabdominal pain or cramping, general well-being, presence ofextraintestinal manifestations of disease, presence of an abdominalmass, use of antidiarrheal drugs, hematocrit, and body weight (Best etal., 1976 Gastroenterology 70: 439-44]; and Summers et al., 1979Gastroenterology 77: 847-69). Scores less than 150 indicated remission;scores between 150 and 219 indicated mildly active disease, between 220and 450 indicated moderately active disease, and scores greater than 450indicated severe activity. Additional prospective endpoints were theproportion of patients demonstrating clinical response (i.e., at least70 point reduction in CDAI), health-related quality of life, as measuredby an inflammatory bowel disease specific questionnaire, the IBDQ(Irvine et al., 1994 Gastroenterology 106: 287-96), and serum levels ofC-reactive protein.

Safety evaluations including the recording of adverse events andmonitoring of clinical laboratories were conducted throughout the study.An independent safety data monitoring committee provided an additionallevel of monitoring. Serum samples were collected at each visit andanalyzed for antibodies against natalizumab by an enzyme linkedimmunosorbent assay (ELISA).

Statistical Analysis

All efficacy analyses utilized the intent-to-treat (ITT) lastobservation carried forward (LOCF) population, which comprised allpatients randomized (n=248). Patients using rescue medications wereclassified as treatment failures. The safety population (n=244)comprised those randomized and dosed. Four patients were not dosed dueto ineligibility.

All statistical tests were two-sided and at the 5 percent level ofstatistical significance. Three pair-wise tests of each active treatmentcompared to placebo were performed. Remission and response rates wereanalyzed by the Cochran Mantel-Haenszel chi-squared test (generalassociation) (Landis et al., 1978 Int'l. Stat. Rev. 46: 237-54) usingcountry as strata. The effect of covariates on remission and response(yes or no) was analyzed at week 6 using logistic regression.

The sample size of 60 subjects per group was calculated to provide 80percent power at a 5 percent significance level to detect a differencein response rates assuming a 40 percent response rate in the natalizumabgroups and a 15 percent response rate in the placebo group.

Two-way analysis of variance models with fixed effects for country andtreatment group were used to compare the mean CDAI decreases frombase-line. Contrasts comparing each of the active treatment groups tothe placebo group were tested.

C-reactive protein and IBDQ data were analyzed using theWilcoxon-Mann-Whitney test to compare the change from base-line betweeneach of the three active treatment groups and placebo. In aprospectively planned analysis, the C-reactive protein levels werecompared for patients with a value above the upper limit of the normalrange of 8 mg/l at base-line (week 0).

Results

Demographic characteristics, CDAI scores, site of disease, andmedications were comparable among the groups at base-line (Table 4). Atthe time of the week 0 assessment, most patients were receiving othermedications for CD including 5-ASA compounds (48 to 64 percent), oralsteroids (46 to 63 percent), or azathioprine/6-mercaptopurine (18 to 37percent) with or without other agents. Twenty-seven patients withdrewfrom the study prior to completing 12 weeks: 10 in the placebo group,and 6, 5, and 6 in the 3+0, 3+3, and 6+6 mg/kg natalizumab groups,respectively.

TABLE 4 DEMOGRAPHIC CHARACTERISTICS AND MEDICATIONS AT BASE-LINECHARACTERISTIC PLACEBO 3 + 0 MG/KG 3 + 3 MG/KG 6 + 6 MG/KG Patientsrandomized 63 68 66 51 Mean age and range (yr)   34 (18-68)   36 (18-66)  36 (19-64)   35 (19-62) Mean disease duration and range (yr)  8.9(0.3-64.3)  8.4 (0.5-27.5)  8.1 (0.5-22)  7.8 (0.6-29) Mean base-lineCDAI and range*  300 (186-447)  288 (211-427)  298 (219-442)  296(210-429) Disease site: Ileal   15 (24%)    9 (13%)   17 (26%)   12(24%) Colonic   11 (17%)   16 (24%)   16 (24%)   16 (31%) Ileocolonic  37 (59%)   43 (63%)   33 (50%)   23 (45%) Gender: Female   33 (52%)  41 (60%)   36 (55%)   26 (51%) Mean weight and range(kg)   68 (42-100)  66 (41-95)   64 (44-97)   69 (44-98) Concomitant Medications Noconcomitant medications* for   12 (19%)   11 (16%)    9 (14%)    5 (10%)Crohn's disease 5 ASA compounds   30 (48%)   40 (59%)   42 (64%)   30(59%) Oral steroids   31 (49%)   31 (46%)   37 (56%)   32 (63%)Azathioprine or 6-MP (±steroids)    2 (35%)   25 (37%)   17 (26%)    9(18%) *Eight of 248 patients had a CDAI score < 220 at base-line. Fiveof these 8 patients were eligible (CDAI ≥ 220) on the basis of theirscreening hematocrit, which was the value available at randomization,but had scores < 220 when subsequently recalculated using the base-linehematocrit. The CDAI scores for the other 3 patients were incorrectlycalculated at the time of randomization. *5 ASAs, steroids, orimmunosuppressants.Clinical Responses, Remissions, and Mean CDAI Scores

The proportion of patients achieving a clinical response (i.e., at leasta 70 point reduction in CDAI from base-line) was statisticallysignificantly greater than placebo in all three natalizumab groups atweeks 4, 6, and 8 (Table 5 and FIG. 6), and this effect persistedthrough week 12 in the two groups that received 2 infusions ofnatalizumab. Trends for improvement in clinical response rates wereobserved as early as 2 weeks after the first treatment, and the 3+3mg/kg natalizumab group demonstrated a statistically significantdifference from placebo at this time point. Results for the decreasefrom base-line in mean CDAI score (Table 6) agree with the response ratefindings.

TABLE 5 RATES OF REMISSION AND CLINICAL RESPONSE IN THE ITT POPULATION3 + 0 MG/KG 3 + 3 MG/KG 6 + 6 MG/KG NATA- NATA- NATA- TIME PLACEBOLIZUMAB LIZUMAB LIZUMAB POINT (N = 63) (N = 68) (N = 66) (N = 51)REMISSION (CDAI < 150) No. OF PATIENTS (%) Week 2  6 (10) 10 (15) 13(20)  6 (12) P value 0.328 0.127 0.745 Week 4  9 (14) 21 (31) 19 (29) 15(29) P value 0.02 0.027 0.028 Week 6 17 (27) 20 (29) 29 (44) 16 (31)(prim. endpoint) P value 0.757 0.030 0.533 Week 8 10 (16) 19 (28) 27(41) 22 (43) P value 0.107 <0.001 <0.001 Week 12 17 (27) 19 (28) 28 (42)21 (41) P value 0.992 0.042 0.091 RESPONSE (≥70 POINT DROP) No. OFPATIENTS (%) Week 2 19 (30) 31 (46) 36 (55) 22 (43) P value 0.081 0.0040.136 Week 4 18 (29) 32 (47) 41 (62) 27 (53) P value 0.029 <0.001 0.006Week 6 24 (38) 40 (59) 47 (71) 29 (57) P value 0.022 <0.001 0.039 Week 822 (35) 38 (56) 44 (67) 28 (55) P value 0.018 <0.001 0.028 Week 12 27(43) 34 (50) 40 (61) 33 (65) P value 0.503 0.033 0.018 Bold highlightsthe statistically significant results when compared to placebo.

TABLE 6 DECREASE FROM BASE-LINE IN MEAN CDAI SCORE 3 + 0 3 + 3 6 + 6MG/KG MG/KG MG/KG NATA- NATA- NATA- TIME PLACEBO LIZUMAB LIZUMAB LIZUMABPOINT (N = 63) (N = 68) (N = 66) (N = 51) MEAN DECREASE IN CDAI SCOREFROM BASE-LINE (SD) Week 2 39.3 (73.5) 63.5 (74.8)  80.0 (68.9)  60.5(68.3)  P value vs. 0.061 0.001 0.103 placebo Week 4 37.3 (88.8) 79.9(89.5) 100.7 (76.4)  84.2 (90.5)  P value vs. 0.004 <0.001 0.003 placeboWeek 6 49.3 (97.8) 81.5 (86.1) 119.4 (79.1)  97.2 (94)   P value vs.0.042 <0.001 0.004 placebo Week 8 49.7 (99.5) 82.4 (87.2) 117.8 (90.7)106.9 (102.9) P value vs. 0.053 <0.001 0.001 placebo Week 12  63.1(103.9) 70.1 (91.7) 119.2 (111)  112.5 (96.4) P value vs. 0.729 0.0010.008 placebo Decrease from baseline is defined as (week 0-week n) SD =standard deviation Bold highlights the statistically significant resultswhen compared to placebo.

Four weeks after the first treatment, and before patients received thesecond treatment, all three natalizumab groups had a statisticallysignificantly higher rate of remission compared to patients in theplacebo group (Table 5 and FIG. 7). However, at week 6, theprospectively defined primary endpoint, the proportion of patients inclinical remission, was statistically significantly higher only in the3+3 mg/kg natalizumab group compared to placebo. At week 8, both groupsthat received two infusions of natalizumab (either 3 or 6 mg/kg)demonstrated statistically significantly higher remission rates comparedto placebo. At week 12, the 3+3 mg/kg natalizumab group continued todemonstrate a statistically significant benefit over placebo forclinical remission, while the 6+6 mg/kg group demonstrated a strongtrend for benefit over placebo for this outcome.

Predictors of Remission or Response

Using the results at week 6, an analysis was performed to identifybase-line variables that may predict remission or clinical response. Thevariables examined included site of disease, duration of disease,base-line CDAI scores, concomitant use of oral steroids, concomitant useof azathioprine or 6-mercaptopurine, and extraintestinal symptoms.Base-line CDAI score was a significant predictor of remission (P<0.001);patients with a higher base-line CDAI were less likely to achieveremission. However, base-line CDAI did not predict the likelihood ofresponse. All other variables analyzed were not significant predictorsof remission or response.

Quality of Life

A statistically significant improvement in mean IBDQ scores was observedin all natalizumab treatment groups at week 6 compared to placebo. Byweek 12, only the treatment groups that received two infusions ofnatalizumab continued to have IBDQ scores that were significantly higherthan the placebo group (Table 7).

TABLE 7 MEAN IBDQ SCORES 3 + 0 MG/KG 3 + 3 MG/KG 6 + 6 MG/KG NATA- NATA-NATA- TIME PLACEBO LIZUMAB LIZUMAB LIZUMAB POINT (N = 63) (N = 68) (N =66) (N = 51) IBDQ SCORE, MEAN (RANGE) Week 0 130 130 (52-188) 136(79-194) 123 (55-194) (66-192) Week 6 142 157 (81-221) 163 (99-211) 155(67-224) P value (61-219) 0.008 <0.001 <0.001 Week 12 145 151 (81-221)163 (86-221) 153 (64-215) P value (61-217) 0.486   0.021   0.014 Boldhighlights improvements in the IBDQ score when compared to eachtreatment base-line.C-Reactive Protein

Patients who had elevated levels of serum C-reactive protein atbase-line received periodic assessments of those levels during thetrial. Those who received two infusions of natalizumab exhibited asignificant decline from base-line in mean serum levels of C-reactiveprotein compared to patients in the placebo group (FIG. 8). Theimprovement was maintained through week 12 in patients who received twoinfusions of 3 mg/kg natalizumab.

Safety and Tolerability

Natalizumab treatment at all dose levels was well tolerated throughoutthe 12-week study period. Similar numbers of patients from each groupwithdrew from the study for adverse events (i.e., 2 in the placebogroup, 1 in the 3 mg/kg natalizumab group, 2 in the 3+3 mg/kgnatalizumab group, and 3 in the 6+6 mg/kg natalizumab group). Overall,31 patients reported a serious adverse event during the main study phase(i.e., 9 in the placebo group, 8 in the 3 mg/kg natalizumab group, 8 inthe 3+3 mg/kg natalizumab group, and 6 in the 6+6 mg/kg natalizumabgroup). No serious adverse events were assessed as related to study drugand none were fatal; the majority were hospital admissions forcomplications or symptoms associated with CD. The numbers of patientsexperiencing treatment-emergent adverse events were similar in thevarious treatment groups: 52 (83 percent) in the placebo group, 51 (78percent) in the 3 mg/kg natalizumab group, 57 (88 percent) in the 3+3mg/kg natalizumab group, and 41 (80 percent) in the 6+6 mg/kgnatalizumab group. Table 8 shows adverse events not related to CD withan incidence at least 5 percent greater in a natalizumab group comparedto the placebo group. None of these various types of adverse events werestatistically significantly different from placebo, and none are deemedto present an unacceptable safety profile for the use of natalizumab inpatients with moderate to severe CD.

TABLE 8 ADVERSE EVENTS NOT RELATED TO CROHN'S DISEASE: WITH > 5%INCIDENCE OVER PLACEBO AND OTHER EVENTS OF INTEREST PLACEBO 3 + 0 3 + 36 + 6 PREFERRED TERM N = 63(%) N = 65 N = 65 N = 51 Chest pain 0 (0) 2(3) 2 (3) 4 (8) Conjunctivitis 0 (0) 2 (3) 5 (8) 0 (0) Dizziness 0 (0) 6(9) 3 (5) 0 (0) Fever 1 (2) 4 (6) 3 (5) 4 (8) Flu syndrome  6 (10)  9(14)  7 (11) 10 (20) Headache 20 (32) 19 (29) 27 (42) 14 (27) Pain 4 (6)4 (6) 4 (6) 11 (22) Sinusitis 0 (0) 0 (0) 0 (0) 3 (6) OTHER EVENTS:Infusion reaction* 0 (0) 0 (0) 1 (2) 1 (2) Pts with anti- 0 (0)  8 (13)4 (6) 1 (2) natalizumab antibodies *Leading to interruption or cessationof infusion.

Anti-natalizumab antibodies were detected in 13 natalizumab-treatedpatients (7 percent) at week 12. Overall, patients with detectableanti-natalizumab antibodies were no more likely to experience an adverseevent or serious adverse event than those who had no detectableanti-natalizumab antibodies.

Two patients experienced infusion reactions; in both, the event occurredduring the second infusion. A patient in the 3+3 mg/kg natalizumab groupexperienced symptoms of mild itching and erythema, and was subsequentlyfound to be positive for anti-natalizumab antibodies. A patient in the6+6 mg/kg natalizumab group experienced symptoms of mild itching andcoughing. These symptoms resolved without treatment, and the patient wassubsequently found to be negative for detectable anti-natalizumabantibodies.

Although the prospectively defined primary efficacy endpoint (clinicalremission defined as CDAI<150 at week 6) was statistically significantlysuperior to placebo only for the 3+3 mg/kg natalizumab group,significant benefit over placebo was observed for this outcome at 4separate time points for the 3+3 mg/kg group (weeks 4, 6, 8, and 12) andtwo time points for the 6+6 mg/kg group (weeks 4 and 8, with a trend forbenefit at week 12). Combined with the findings that the proportions ofpatients experiencing a clinical response (defined as at least a 70point drop in CDAI from base-line) were significantly superior toplacebo for all three natalizumab groups at the week 4, 6, and 8 timepoints, and at the week 12 time point for the two treatment groups thatreceived two infusions of natalizumab, these data provide strongevidence for efficacy of natalizumab in the treatment of moderate toseverely active CD. Further, the beneficial effects of natalizumab onclinical response and remission rates based on improvements in the CDAIare corroborated by significant improvements in health related QOL,measured by the IBDQ, and improvement in serum C-reactive protein, anacute phase reactant used to quantify generalized inflammation.

The dosage levels of receptor saturation in the CD trial are comparableto the dosages of the MS trial, and thus receptor saturation levelsshould be comparable in the CD trial. Receptor saturation levels in theCD trial are thus associated with decreased levels of inflammation, asdemonstrated by C-reactive protein levels and an improvement in overallpatient well-being, as demonstrated by the CDAI.

No serious safety concerns were identified for any of the natalizumabtreatment groups. The percentage of patients developing detectableanti-natalizumab antibodies was low, and there were no serious adverseevents associated with detectable anti-natalizumab antibodies.

The present study provides compelling evidence for the effectiveness andtolerability of alpha-4 integrin antagonism by natalizumab in thetreatment of the clinical signs and symptoms of active moderate tosevere CD. In addition, the evidence these data provide that alpha-4integrin antagonism is an effective mechanism for treating CD raises thepossibility that this modality may be more broadly applicable to thetreatment of chronic autoimmune and inflammatory diseases.

Example 3 The Effect of Natalizumab on Circulating Activated Leucocytesin Active Inflammatory Bowel Disease

The trafficking of leukocyte subsets is involved in the pathogenesis ofinflammatory bowel disease (IBD). Because alpha-4 integrins are keymediators of leukocyte migration across the vascular endothelium, beingthat they are expressed on all leucocytes except neutrophils, theeffects of a single 3 mg/kg natalizumab (Antegren®) infusion on basiccirculating leukocyte subsets, natural killer (NK) cells and activated Tcells in active inflammatory bowel disease patients (IBD). It had alsopreviously been shown that a single 3 mg/kg infusion of natalizumabproduced a sustained rise in circulating peripheral blood leucocytes inanimals and healthy volunteers (“A six-month weekly intravenous toxicitystudy with Antegren™ in cynomolgous monkeys with a six-week recovery,”Athena Report 1998, No. 723-013-098). However, it was unknown whethernatalizumab could have differential effects on leukocyte subsets, giventhat all leucocytes except neutrophils express alpha-4 integrins.

Methods. Leucocytes extracted from peripheral blood of 30 Crohn'sdisease (CD; 18 natalizumab, 12 placebo) and 10 ulcerative colitis (UC;all received natalizumab—no placebo) patients pre-, 1-, 2-, 4-, 8- and12-weeks post-infusion were analyzed by fluorescence-activatedcell-sorter (FACS). Serum natalizumab levels and disease activity scoreswere measured at each time-point.

Significant changes compared to baseline (p<0.05) and correlations weretested by Wilcoxon and Spearman tests respectively. Wilcoxon signed ranktest for analysis of changes in leukocyte subsets were compared tobaseline within treatment groups (p<0.05 denotes significance). Spearmanrank correlation tests were used to assess correlation between diseaseactivity parameters, natalizumab levels and leukocyte subsets in thosepatients receiving natalizumab.

Venous blood was taken immediately prior to natalizumab/placebo infusionand again at one, two, four, eight and twelve weeks post-infusion. TheLymphoprep™ method (Nycomed, Denmark) was used to isolate the peripheralblood lymphocytes (PBLs) prior to analysis by multi-colorfluorescence-activated cell sorter (FACS; Becton Dickenson, Oxford, UK)in conjunction with Consort 30 software (Amlot et al., 1996 Clin. Exp.Immunol. 105: 176-82). The percentages of PBLs expressing the followingmarkers were measured using FACS analysis: CD I9 (B cell), TCRαβ (Tcell), CD3 (pan-T cell), TCRγδ (T cell), CD4 (helper/Th-1 T cell), CD8(cytotoxic/suppressor T cell) and CD16 (NK cell). The percentages ofTCRαβ cells expressing the activation antigens CD38, CD25 chain ofinterleukin-2 receptor), CD26, CD69 and HLA-DR were measured, inaddition to naïve (CD45RA) and memory (CD45RO) T cell subsets and “NK-Tcells” (CD57⁺/CD3⁺). The percentage of cytotoxic/suppressor T cells(CD8⁺) expressing the activation antigens CD28 and HLA-DR were alsomeasured.

Results. Eosinophil, monocyte, B and T cell counts were allsignificantly raised for ≥1 week post-natalizumab. Total lymphocytecounts, both B and T cells, were significantly increased as compared tobaseline levels. Neutrophil and basophil counts were unchanged. T cellsexpressing the activation markers CD25, CD26, HLA-DR, CD8DR, CD8, CD28,CD45RO and CD45RA were significantly raised compared to baseline at ≥4and 1 week in UC and CD patients respectively. CD38⁺ and CD69⁺ T cellswere raised at ≥1 week in UC patients only. NK cells were unchangedpost-infusion in all patients and NK-type T cells (CD57⁺) were raised at1 week in CD patients only. No significant changes in gamma-delta (γδ) Tcells were found in CD/UC patients. Changes in T cell subsets did notcorrelate with disease activity or serum natalizumab levels. Theleukocyte changes found post-natalizumab were not detected followingplacebo.

The lymphocyte counts remained elevated at 4 weeks post-infusion in bothCrohn's disease patients (p=0.002) and in those patients with ulcerativecolitis (p=0.02), before returning to pre-treatment values at week 8(Gordon et al., 2002 Aliment. Pharm. & Ther. 16: 699-706; and Gordon etal., 2001 Gastroenterology 121: 268-74).

The effect of natalizumab on circulating eosinophils and monocytes areshown in FIGS. 9A and 9B. The neutrophil and basophil counts wereobserved to remain unchanged in all the patient groups studied. FIGS. 10and 11 demonstrate the effects of natalizumab administration on specificcirculating T cell subsets and natural killer cells in Crohn's diseaseand ulcerative colitis patients. The error bars denote standards ofdeviation in each chart.

No significant changes in basic leukocyte subsets were detectedfollowing placebo infusion. Administration of the placebo to the 11placebo patients is shown in Table 9. Some changes in lymphocytesexpressing activation antigens were detected at isolated time pointsonly in Crohn's patients (Table 10).

TABLE 9 LEUKOCYTE SUBSETS IN PLACEBO-TREATED CROHN'S DISEASE PATIENTSSubset Cells × Week 0 Week 1 Week 2 Week 4 Week 8 10⁶/mL Mean SD Mean SDMean SD Mean SD Mean SD Lymphocytes .60 .33 .55 .36 .82 .67 .48 .29 .51.31 Neutrophils .21 .75 .18 .48 .86 .36 .61 .26 .91 .15 Eosinophils .19.16 .17 .13 .15 .13 .20 .12 .14 .10 Basophils .19 .30 .14 .09 .09 .07.09 .03 .08 .05 Monocytes .60 .33 .55 .36 .82 .67 .48 .29 .51 .31 Mean(SD) leukocyte subset values post-placebo; no significant differences inany group compared to baseline (week 0) values.

TABLE 10 T CELL AND NK MARKERS IN PLACEBO- TREATED CROHN'S DISEASEPATIENTS Subset Cells × Week 0 Week 1 Week 2 Week 4 Week 8 Week 1210⁶/mL Mean SD Mean SD Mean SD Mean SD Mean SD Mean SD Lymphocytes .36.57 .12 .57 .83 .08 .34 .98 .28 .74 .62 .84 TCRαβ+ .99 .53 .81 .56 .28.88 .98 .84 .95 .79 .17 .81 CD25+ .18 .11 .12 .07 .27 .22 .17 .17 .18.15 .22 .17 CD26+ .49 .32 .38 .27 .80 .61 .54 .49 .54 .55 .58 .43CD45RA+ .74 .41 .57 .39 .95 .69 .68 .59 .68 .57 .84 .56 CD45RO+ .42 .20.31

.50 .27 .39 .24 .42 .35 .50 .33 CD38+ .42 .29 .31

.46 .38 .35 .27 .33 .25 .40 .25 CD69+ .14 .20 .67 .71 .13 .12 .05 .04.12 .22 .10 .08 HLA-DR+ .19 .10 .15

.25 .20 .16 .11 .17 .10 .22 .16 CD8+ .11 .09 .09

.16 .15 .09 .07 .08 .05 .12 .11 CD8+CD28+ .21 .11 .16 .11 .30 .20 .22.21 .20 .18 .23 .14 CD8+DR+ .11 .09 .09 .06 .16 .15 .09 .07 .08 .05 .12.11 CD57+CD3+ .08 .06 .06 .05 .10 .10 .07

.07 .05 .10 .15 CD16+CD3− .11 .09 .12 .09 .16 .18 .09 .04 .12 .12 .17.19 TCRγδ+ .11 .16 .08 .09 .18 .28 .10 .13 .09 .09 .11 .10 KappaMAb+ .02.02 .03 .05 .05 .08 .07 .09 .03

.10 .01 Columns show mean counts (SD) of T cell subsets and NK-typecells of Crohn's study placebo patients compared with baseline values(Wilcoxon signed rank test; p < 0.05). Significant differences comparedto baseline are shown in bold. Standard deviations for each mean areshown in italic numbers.

There was no significant correlation between the total lymphocyte countsand disease activity score in either Crohn's disease or ulcerativecolitis patients (Tables 11 and 12), nor were any significantcorrelations found between individual lymphocyte subsets and diseaseactivity. No significant correlation was detected between serumnatalizumab levels and changes in leukocyte subsets at one, two or fourweeks post-infusion. Natalizumab was undetectable in almost all patientsat eight weeks. Thus correlations were not calculated for this timepoint.

TABLE 11 SPEARMAN R VALUES COMPARING LYMPHOCYTE SUBSETS AND DISEASEACTIVITY Crohn's disease Ulcerative colitis Subsets Week 1 Week 2 Week 4Week 1 Week 2 Week 4 Lymphocytes 0.16 −0.37 −0.01 −0.63 −0.19 0.03 TCRαβ0.26 −0.29 0.05 −0.59 −0.39 0 CD25 0.44 0.07 0.13 −0.39 −0.46 0.17 CD260.31 −0.22 −0.11 −0.39 −0.29 0.10 CD45RA 0.29 −0.25 0.19 −0.54 −0.390.51 CD45RO 0.32 −0.27 0.34 0.18 −0.17 −0.05 CD38 0.31 −0.08 0.01 −0.570.03 −0.05 CD69 −0.32 0.12 0.12 −0.15 0.28 0.12 HLA-DR 0.19 −0.05 −0.010.12 −0.13 0.15 CD8CD28 0.01 −0.17 −0.13 −0.18 −0.04 −0.17 CD8DR 0.11−0.19 −0.18 −0.1 0.07 0.24 CD57 0.19 −0.08 −0.04 0.21 −0.15 0.15 CD16−0.08 −0.3 −0.15 −0.19 0.1 −0.32 TCRγδ 0.34 0.14 −0.31 −0.63 −0.47 −0.66Kappa Mab −0.11 −0.02 −0.2 0.05 −0.27 0.19 No significant correlationfound between lymphocyte subset counts and CDAI (Crohn's diseasepatients) or Powell-Tuck score (ulcerative colitis).

TABLE 12 SPEARMAN R VALUES COMPARING LYMPHOCYTE SUBSETS WITH SERUMNATALIZUMAB Crohn's disease Ulcerative colitis Subsets Week 1 Week 2Week 4 Week 1 Week 2 Week 4 Lymphocytes 0.03 0.49 0.64 0.1 −0.02 0.18TCRαβ −0.11 0.35 0.51 −0.04 0.28 −0.11 CD25 −0.09 0.03 0.26 0.25 −0.07−0.25 CD26 −0.07 0.28 0.61 0.20 0.22 −0.14 CD45RA 0.19 0.29 0.62 −0.12−0.25 −0.32 CD45RO −0.16 0.32 0.34 0.37 0.08 0.39 CD38 0.06 0.18 −0.34−0.01 −0.45 −0.43 CD69 0.02 0.26 0.57 0.2 −0.22 0.14 HLA-DR 0.05 0.13−0.25 0.08 0.27 −0.07 CD8CD28 −0.18 0.16 0.41 0.25 0.07 0.32 CD8DR 0.40.16 0.004 0.08 0.3 −0.18 CD57 0.1 −0.06 −0.31 −0.08 0.03 0 CD16 0.220.19 −0.5 0.13 0.37 0.25 TCRγδ 0.002 −0.16 −0.2 0.21 0.61 0.6 Kappa MAb−0.07 −0.27 0.29 0.41 0.45 −0.41 No significant correlation betweendisease activity and lymphocyte subsets, except for total lymphocytecounts at week 4 (p = 0.04).

Based on the above data, a single 3 mg/kg natalizumab infusion producedincreased circulating levels of most, but not all, leukocyte subsets inpatients with active IBD. Circulating eosinophil, monocyte andlymphocyte counts were significantly elevated above baseline values forat least four weeks post-infusion in most patients. A wide range ofcirculating T cell subsets were significantly increased abovepretreatment values, particularly those expressing activation antigens.However, NK cell counts (CD16⁺/CD3⁻) were not affected by natalizumab,and CD57⁺ T cells were affected to a much lesser extent by natalizumabthan other T cell subsets. Lymphocytes expressing the γδT cell receptorwere also not affected by natalizumab, suggesting that alpha-4 integrinsare either not expressed, or are expressed at a lower level on thesecells.

Thus, in active IBD patients, natalizumab may limit trafficking andmaintain in circulation many leukocyte and activated lymphocyte subsets.The NK cells, γδ cells, neutrophils and basophils appear unaffected bythe administration of natalizumab, which may suggest that they are lessimportant mediators in the trafficking of these cell types.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe invention.

All references cited herein are herein incorporated by reference intheir entirety for all purposes.

We claim:
 1. A method of monitoring a chronic administration regimen fortreating pathological inflammation in a subject, wherein thepathological inflammation is modulated by an alpha-4 integrin,comprising chronically administering 300 mg natalizumab or animmunologically active fragment thereof by infusion every four weeks tothe patient for a period of at least six months; and detecting levels ofbrain lesions using an imaging technique.
 2. The method of claim 1,wherein the pathological inflammation is caused by multiple sclerosis.3. The method of claim 1, wherein the method comprises chronicallyadministering 300 mg natalizumab.
 4. The method of claim 3, wherein theimaging technique is magnetic resonance imaging (MRI).
 5. The method ofclaim 4, wherein the pathological inflammation is caused by multiplesclerosis.
 6. The method of claim 5, wherein the method comprisesdetecting presence or absence of new lesions in the central nervoussystem using MRI.
 7. The method of claim 5, wherein the method comprisesdetecting Gd-enhancing brain lesions.